Long-term resistance to tomato spotted wilt virus in transgenic tobacco cultivars expressing the viral nucleoprotein gene: greenhouse and field tests

We examined the resistance phenotype of a large number of transgenic tobacco plants originating from 12 commercial (Nicotiana tabacum) cultivars expressing the sense form of the nucleoprotein (N) gene of L3, a Bulgarian isolate of tomato spotted wilt virus (TSWV). The analysis revealed that transgenic plants are completely protected against the homologous L3 isolate of TSWV irrespective of whether or not they contain detectable levels of translational product. The effectiveness of protection against the virus was investigated upon mechanical inoculation under greenhouse conditions and in field trials. Non-segregating resistant lines were selected and the inheritance of the resistance to TSWV was analysed in successive generations (R₃–R₆). Extensive tests under controlled conditions and two-year field trials proved that the resistance to TSWV is stable in different environments and is a stably inherited trait.     

Engineered resistance to tomato spotted wilt virus in transgenic peanut expressing the viral nucleocapsid gene

The nucleocapsid gene of tomato spotted wilt virus Hawaiian L isolate in a sense orientation, and the GUS and NPTII marker genes, were introduced into peanut (Arachis hypogaea cv. New Mexico Valencia A) using Agrobacterium-mediated transformation. Modifications to a previously defined transformation protocol reduced the time required for production of transformed peanut plants. Transgenes were stably integrated into the peanut genome and transmitted to progeny. RNA expression and production of nucleocapsid protein in transgenic peanut were observed. Progeny of transgenic peanut plants expressing the nucleocapsid gene showed a 10- to 15-day delay in symptom development after mechanical inoculations with the donor isolate of tomato spotted wilt virus. All transgenic plants were protected from systemic tomato spotted wilt virus infection. Inoculated non-transformed control plants and plants transformed with a gene cassette not containing the nucleocapsid gene became systemically infected and displayed typical tomato spotted wilt virus symptoms. These results demonstrate that protection against tomato spotted wilt virus can be achieved in transgenic peanut plants by expression of the sense RNA of the tomato spotted wilt virus nucleocapsid gene     

A novel tomato spotted wilt virus isolate encoding a noncanonical NSm C118F substitution associated with Sw-5 tomato gene resistance breaking

The nonstructural protein NSm of tomato spotted wilt virus (TSWV) has been identified as the avirulence determinant of the tomato single dominant Sw-5 resistance gene. Although Sw-5 effectiveness has been shown for most TSWV isolates, the emergence of resistance-breaking (RB) isolates has been observed. It is strongly associated with two point mutations (C118Y or T120N) in the NSm viral protein. TSWV-like symptoms were observed in tomato crop cultivars (+Sw-5) in the Baja California peninsula, Mexico, and molecular methods confirmed the presence of TSWV. Sequence analysis of the NSm 118–120 motif and three-dimensional protein modelling exhibited a noncanonical C118F substitution in seven isolates, suggesting that this substitution could emulate the C118Y-related RB phenotype. Furthermore, phylogenetic and molecular analysis of the full-length genome (TSWV-MX) revealed its reassortment-related evolution and confirmed that putative RB-related features are restricted to the NSm protein. Biological and mutational NSm 118 residue assays in tomato (+Sw-5) confirmed the RB nature of TSWV-MX isolate, and the F118 residue plays a critical role in the RB phenotype. The discovery of a novel TSWV-RB Mexican isolate with the presence of C118F substitution highlights a not previously described viral adaptation in the genus Orthotospovirus, and hence, the necessity of further crop monitoring to alert the establishment of novel RB isolates in cultivated tomatoes.     

接种番茄斑萎病毒番茄植株对西花蓟马生物学特性的影响

西花蓟马Frankliniella occidentalis(Pergande)是我国的一种重要入侵害虫。本文研究了西花蓟马在番茄3种处理(健康CK、机械接种番茄斑萎病毒MI、机械损伤MD)叶片上的生长发育、存活及种群增长。结果表明:健康、机械接种番茄斑萎病毒、机械损伤叶片上的发育历期依次为12.68、12.99和11.79d。雌雄成虫寿命和雌虫繁殖能力在各处理叶片上差异不显著(P>0.05)。健康、机械接种番茄斑萎病毒、机械损伤叶片上的内禀增长率依次为0.1362、0.1526和0.1292d-1。本研究表明,接种番茄斑萎病毒的番茄叶片未缩短西花蓟马发育历期,也不能延长寿命及提高产卵量,不能明显加速种群数量增长。这意味着番茄斑萎病毒对西花蓟马在番茄叶片上的生物学特性未能产生明显的有利作用。     

Selection of resistance breaking strains of tomato spotted wilt tospovirus

In glasshouse tests, sap from plants infected with 15 different isolates of tomato spotted wilt tospovirus (TSWV) from three Australian states was inoculated to nine genotypes of tomato carrying TSWV resistance gene Sw-5 or one of its alleles. A further two resistant tomato genotypes were inoculated with four isolates each. The normal response in resistant genotypes was development of necrotic local lesions in inoculated leaves without systemic invasion, but 22/752 plants also developed systemic reactions in addition to local hypersensitive ones. Using cultures from two of these systemically infected plants and following four cycles of subculture in TSWV resistant tomato plants, two isolates were obtained that gave susceptible type systemic reactions but no necrotic spots in inoculated leaves of resistant tomatoes. When these two isolates, Da_WA-1d and To_TAS-1d, were maintained by repeated subculture for 10 successive cycles in Nicotiana glutinosa or a susceptible tomato genotype, they still induced susceptible type systemic reactions when inoculated to resistant tomato plants. They were therefore stable resistance breaking isolates as regards overcoming gene Sw-5. When resistance-breaking isolate Da_WA^-1ld multiplied together with original isolate Da_WA-l in susceptible tomato, it was fully competitive with the original isolate. However, when Da_WA-ld and To_TAS-ld were inoculated to TSWV resistant Lycopersicon peruvianum lines PI 128660R and PI 128660S and to TSWV resistant Capsicum chinense lines PI 152225, PI 159236 and AVRDC CO0943, they failed to overcome the resistance, producing only necrotic local lesions without systemic infection. Thus, although the ease of selection, stability and competitive ability of resistance breaking isolates of TSWV is cause for concern, L. peruvianum and C. chinense lines are available which are effective against them. The effectiveness of the resistance to TSWV in nine tomato genotypes was examined in a field experiment. Spread was substantial in the susceptible control genotype infecting 42% of plants. Resistance was ineffective in cv. Bronze Rebel, 26% of plants developing infection. In contrast, it held up well in the other eight resistant genotypes with only 1–3 or no plants of each becoming infected. Accumulated numbers of Thrips tabaci, Frankliniella occidentalis and F. schultzei were closely correlated with TSWV spread.     

寄主植物接种番茄斑萎病毒对西花蓟马种群的影响

【目的】西花蓟马Frankliniella occidentalis (Pergande)是一种入侵我国的重要害虫, 而番茄斑萎病毒是以西花蓟马传播为主的一种极具危害性的世界性病毒, 通过研究西花蓟马与番茄斑萎病毒之间的互作将有助于进一步深入理解西花蓟马以及番茄斑萎病毒的发生与猖獗机制, 同时也将为制定合理、可持续的控制西花蓟马及其传播的植物病毒防控策略提供理论依据。【方法】利用应用特定年龄-龄期及两性生命表方法, 研究了西花蓟马在辣椒3种处理（健康CK、机械损伤MD、机械接种番茄斑萎病毒MI）叶片上的生长发育、存活及种群增长。【结果】健康、机械损伤和机械接毒叶片上的发育历期依次为12.45, 11.97和11.18 d。雌雄成虫寿命和雌虫产卵量在不同处理植株叶片上差异显著(P<0.05), 在机械接毒叶片上寿命最长(雌13.51 d, 雄12.69 d); 繁殖能力最强, 产子代数高达33.01头1龄若虫/雌。健康、机械损伤和机械接毒叶片上西花蓟马内禀增长率分别为-0.009, 0.153和0.190 d-1, 净生殖率依次为0.84, 14.54和21.79。【结论】番茄斑萎病毒诱导寄主植物辣椒反应使西花蓟马发育历期缩短, 成虫寿命延长, 繁殖能力提高, 种群增长加速。     

转基因植物中RNA介导的病毒抗性研究进展

利用病毒核酸序列培育抗病毒的转基因植物是一个重要的抗病毒基因工程策略。虽然很多种病毒的不同核酸序列已被使用并证明转基因植物有不同程度的抗病毒效果，但其抗病机制大多不清楚。目前至少有两种明显不同的抗病机制类型：一种是要求病毒编码的蛋白质的表达；另一种是仅仅依靠转基因的ＴＮＡ转录。本文综述了这种ＲＮＡ介导的抗性特点、分子生物学、抗病机制，以及与共抑制的相似性，并对ＲＮＡ介导的病毒抗性的意义加以讨论。     

Extreme resistance to cucumber mosaic virus (CMV) in transgenic tomato expressing one or two viral coat proteins

For the production of broad commercial resistance to cucumber mosaic virus (CMV) infection, tomato plants were transformed with a combination of two coat protein (CP) genes, representing both subgroups of CMV. The CP genes were cloned from the CMV-D strain and Italian CMV isolates (CMV-22 of subgroup I and CMV-PG of subgroup II) which have been shown to produce severe disease symptoms. Four plant transformation vectors were constructed: pMON18774 and pMON18775 (CMV-D CP), pMON18831 (CMV-PG CP) and pMON18833 (CMV-22 CP and CMV-PG CP). Transformed R0 plants were produced and lines were selected based on the combination of three traits: CMV CP expression at the R0 stage, resistance to CMV (subgroup I and/or II) infection in growth chamber tests in R1 expressing plants, and single transgene copy, based on R1 segregation. The results indicate that all four vector constructs generated plants with extremely high resistant to CMV infection. The single and double gene vector construct produced plants with broad resistance against strains of CMV from both subgroups I and II at high frequency. The engineered resistance is of practical value and will be applied for major Italian tomato varieties. This revised version was published online in June 2006 with corrections to the Cover Date.     

Occurrence and localization of spherical viruslike particles in tissues of apparently healthy tobacco thrips,Frankliniella fusca,a vector of tomato spotted wilt virus

Spherical viruslike particles (VLP) were found in the tissues of apparently healthy tobacco thrips, Frankliniella fusca. The particles occurred in abundance in thrips from Ontario but were absent in thrips from Oklahoma reared under identical conditions. The VLP were not transmissible to any of the seven plant hosts (in four families) of F. fusca suggesting that they may be an insect virus. Transmission of tomato spotted wilt virus (TSWV) by F. fusca, a known vector, was not affected by the presence or absence of the VLP. No TSWV particles were detected in tissues of F. fusca that transmitted TSWV to test plants. The VLP occurred in several internal organs and hemocoele of the thrips and were isolated in vitro by preparing homogenates of gut tissues. Infection of oocytes and presence of VLP in young nymphs suggested transovarial transmission of the particles. The VLP measured 62 ± 4 nm in diameter and usually occurred in dense viroplasms in the cell cytoplasm. Development of the particles within the viroplasms is discussed.     

Evaluation of resistance in Osteospermum ecklonis (DC.) Norl. plants transgenic for the N protein gene of tomato spotted wilt virus

 The nucleocapsid protein (N) gene of tomato spotted wilt virus (TSWV) was inserted into Osteospermum ecklonis via Agrobacterium tumefaciens leaf strips co-cultivation. Sixteen primary transformant clones of two O. ecklonis genotypes were analysed. Southern blots of restricted genomic DNA demonstrated integration of the transgene and indicated the number of integrated copies. Expression of the transgene was estimated by DAS-ELISA and Western and Northern blotting. Plants were challenged with TSWV inoculation, either mechanically or by the thrips Frankliniella occidentalis; they were then monitored for symptom appearance and tested by TAS-ELISA for infection. Inoculation of the transgenic clones via the natural TSWV vector was more efficient and led to the identification of 1 clone, characterised by multiple transgene integration and no transgene expression, with improved resistance to TSWV. Received: 20 November 1999 / Revision received: 11 February 2000 / Accepted: 22 February 2000     

Transgenic peanut plants containing a nucleocapsid protein gene of tomato spotted wilt virus show divergent levels of gene expression

The nucleocapsid protein (N) gene of the lettuce isolate of tomato spotted wilt virus (TSWV) was inserted into peanut (Arachis hypogaea L.) via microprojectile bombardment. Constructs containing the hph gene for resistance to the antibiotic hygromycin and the TSWV N gene were used for bombardment of peanut somatic embryos. High frequencies of transformation and regeneration of plants containing the N gene were obtained. Southern blot analysis of independent transgenic lines revealed that one to several copies of the N gene were integrated into the peanut genome. Northern blot, RT-PCR and ELISA analyses indicated that a gene silencing mechanism may be operating in primary transgenic lines containing multiple copy insertions of the N transgene. One transgenic plant which contained a single copy of the transgene expressed the N protein in the primary transformant, and the progeny segregated in a 3 :1 ratio based upon ELISA determination. Received: 24 October 1997 / Revision received: 9 February 1998 / Accepted: 21 February 1998     

Characterization of cymbidium mosaic virus coat protein gene and its expression in transgenic tobacco plants

Cymbidium mosaic virus (CyMV) is the most prevalent virus infecting orchids. Here, we report the isolation of partial cDNA clones encoding the genomic RNA of CyMV. Like most of the polyadenylated monopartite positive-strand RNA viruses, the open reading frame (ORF) coding for the viral coat protein (CP) is located at the 3 end. The ORF predicts a polypeptide chain of 220 amino acids with a molecular weight of 23 600. Sequence comparison of this ORF to the CP sequences of potato virus X(PVX) and white clover mosaic virus (WCIMV) revealed a strong amino acid homology in the mid-portion of the CP, but the overall homology was low. The CyMV CP gene was placed downstream of a cauliflower mosaic virus 35S promoter and the chimaeric gene was transferred into Nicotiana benthamiana. Transgenic plants expressing the CyMV CP were protected against CyMV infection.     

Identification of new sources of resistance to resistance-breaking isolates of tomato spotted wilt virus

The tomato as both a fresh consumption and industrial product is one of the most profitable vegetables and has a large cultivation area in the world. Parallel to intense production activities, Tomato Spotted Wilt Virus (TSWV), like viral diseases, results in significant economic losses every year. Use of resistant cultivars is the most efficient and environmental-friendly method of fighting against these diseases. This study was conducted to develop new tomato genetic resources resistant to TSWV because of the Sw-5 resistance breaking (RB) isolates that were determined in tomato cultivation areas. In this study, a total of 40 tomato materials including 15 lines, 9 commercial varieties and 16 wild genotypes were by tested with molecular and biological testing methods. Mechanical inoculation method was used for biological testing and SCAR marker was used in molecular analysis. S. penellii, S. chmielewski, S. habrochaites, S. peruvianum and S. sitiens, LA0716, LA1028, LA1777, LA2744 and LA4110 genotypes were found as resistant against breaking isolates of Tomato Spotted Wilt Virus. These genotypes may be a good resistance source for the future breeding studies in tomato.     

Propagation of tomato spotted wilt virus in Frankliniella occidentalis does neither result in pathological effects nor in transovarial passage of the virus

The effect of tomato spotted wilt virus (TSWV) on Frankliniella occidentalis Pergande (Thysanoptera; Thripidae) following a 6-hour acquisition access period on infected plants was investigated. No statistically significant differences were observed among viruliferous, non-viruliferous and control thrips with respect to developmental time, reproduction rate and survival. Thrips larvae, exposed or non-exposed to TSWV, developed from egg to adult in 13.1 and 13.2 days, respectively. Exposed females produced an average of 28.3 larvae whereas control thrips produced 22.3 larvae and longevity was 13.4 and 12.5 days, respectively. None of these values were significantly different. Population reproductive statistics, net reproductive rate (R ₀), mean generation time (T) and intrinsic rate of increase (r _m) were calculated from the life fertility tables. R ₀ and r _m were higher for viruliferous thrips as compared to non-viruliferous and non-exposed thrips. Virus transmission studies revealed that viruliferous thrips were able to transmit virus until death and that TSWV was not transovarially transmitted.     

Protection against virus infection in tobacco plants expressing the coat protein of grapevine fanleaf nepovirus

Summary Grapevine fanleaf nepovirus (GFLV) is responsible for the economically significant court-noué disease in vineyards. Its genome is made up of two single-stranded RNA molecules (RNA1 and RNA2) which direct the synthesis of polyproteins P1 and P2 respectively. A chimeric coat protein gene derived from the C-terminal part of P2 was constructed and subsequently introduced into a binary transformation vector. Transgenic Nicotiana benthamiana plants expressing the coat protein under the control of the CaMV 35S promoter were engineered by Agrobacterium tumefaciens-mediated transformation. Protection against infection with virions or viral RNA was tested in coat protein-expressing plants. A significant delay of systemic invasion was observed in transgenic plants inoculated with virus compared to control plants. This effect was also observed when plants were inoculated with viral RNA. No coat protein-mediated cross-protection was observed when transgenic plants were infected with arabis mosaic virus (ArMV), a closely related nepovirus also responsible for a court-noué disease.Abbreviations GFLV-F13 grapevine fanleaf virus F13 isolate - ArMV arabis mosaic virus - CP coat protein - MS Murashige and Skoog - NPTII neomycin phosphotransferase II - CaMV cauliflower mosaic virus - ELISA enzyme linked immunosorbent assay - VPg genome linked viral protein - TMV tobacco mosaic virus - PVX potato virus X - PVY potato virus Y - TRV tobacco rattle virus - +CP CP expressing - -CP control plant, not expressing CP - CPMP coat protein-mediated protection - CPMCP coat crotein-mediated cross protection     

Paraquat resistance of transgenic tobacco plants over-expressing the Ochrobactrum anthropi pqrA gene

Transgenic tobacco plants over-expressing the Ochrobactrum anthropi pqrA gene, which encodes a membrane transporter mediating resistance to paraquat, were generated. Transgenic plants displayed higher resistance against paraquat than wild-type plants, as estimated by plant viability, ion leakage and chlorophyll loss, but no resistance against other active oxygen generators, such as H2O2 and menadione. Moreover, lower levels of paraquat accumulated in transgenic plants, compared to wild-type plants, indicating that the PqrA protein detoxifies paraquat either via increased efflux or decreased uptake of the herbicide, but not by removing active oxygen species. The results collectively demonstrate that the bacterial paraquat resistance gene, pqrA, can be functionally expressed in plant cells, and utilized for the development of paraquat-resistant crop plants.     

Resistance to tomato spotted wilt virus infection in transgenic tobacco expressing the viral nucleocapsid gene.

A recombinant plasmid containing the entire tomato spotted with virus (TSWV) nucleocapsid gene, with the exception of nucleotide encoding three N-terminal amino acids, was isolated by screening a complementary DNA library, prepared against random primed viral RNA, using a specific monoclonal antibody. The insert contained in plasmid pTSW1 was repaired and amplified by polymerase chain reaction, and the complete nucleocapsid protein gene was introduced into Nicotiana tabacum 'Samsun' by leaf disk transformation using Agrobacterium tumefaciens. Transgenic plants expressing the viral nucleocapsid protein were resistant to subsequent infection following mechanical inoculation with TSWV as indicated by a lack of systemic symptoms and little or no systemic accumulation of virus as determined by double antibody sandwich enzyme-liked immunosorbent assay. These results further extend the applicability of coat protein-mediated resistance, as previously demonstrated for a number of simple plant viruses composed of a positive-sense RNA genome encapsidated with a single species of coat protein, to a membrane-encapsidated, multi-component, negative-sense RNA virus.     

Expression of a bacterial gene in transgenic tobacco plants confers resistance to the herbicide 2,4-dichlorophenoxyacetic acid

Plants resistant to the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) were produced through the genetic engineering of a novel detoxification pathway into the cells of a species normally sensitive to 2,4-D. We cloned the gene for 2,4-D monooxygenase, the first enzyme in the plasmid-encoded 2,4-D degradative pathway of the bacterium Alcaligenes eutrophus, into a cauliflower mosaic virus 35S promoter expression vector and introduced it into tobacco plants by Agrobacterium-mediated transformation. Transgenic tobacco plants expressing the highest levels of the monooxygenase enzyme exhibited increased tolerance to 2,4-D in leaf disc and seed germination assays, and young plants survived spraying with levels of herbicide up to eight times the usual field application rate. The introduction of the gene for 2,4-D monooxygenase into broad-leaved crop plants, such as cotton, should eventually allow 2,4-D to be used as an inexpensive post-emergence herbicide on economically important dicot crops.     

Transgenic tobacco plants expressing the coat protein of cucumber mosaic virus show different virus resistance

Transgenic tobacco (Nicotiana tabacum cv. Xanthi-nc) plants were regenerated after cocultivation of leaf explants withAgrobacterium tumefaciens strain LBA4404 harboring a plasmid that contained the coat protein (CP) gene of cucumber mosaic virus (CMV-As). PCR and Southern blot analyses revealed that the CMV CP gene was successfully introduced into the genomic DNA of the transgenic tobacco plants. Transgenic plants (CP⁺) expressing CP were obtained and used for screening the virus resistance. They could be categorized into three types after inoculation with the virus: virus-resistant, delay of symptom development, and susceptible type. Most of the CP⁺ transgenic tobacco plants failed to develop symptoms or showed systemic symptom development delayed for 5 to 42 days as compared to those of nontransgenic control plants after challenged with the same virus. However, some CP⁺ transgenic plants were highly susceptible after inoculation with the virus. Our results suggest that the CP-mediated viral resistance is readily applicable to CMV disease in other crops.