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     

A multi-generation analysis of the stability of transgenic virus resistance in doubled-haploid tobacco lines

Plants can be genetically engineered for virus resistance by transformation with a viral gene. We transformed tobacco with the tomato spotted wilt virus (TSWV) nucleocapsid gene from the Hawaiian L isolate in order to obtain TSWV resistant breeding lines. Doubled-haploid lines were produced from primary transgenic plants that were selected for resistance to the virus. Several of these lines showed very high levels of resistance and were symptomless after inoculation with the Hawaiian L isolate of TSWV. The accumulation of only low levels of full-length transgene RNA and protein observed in these lines is consistent with an RNA-mediated mechanism of resistance. The lines that were highly resistant to the Hawaiian L isolate of TSWV were also found to be highly resistant to several other isolates of TSWV, while lines that were only moderately resistant to the Hawaiian L isolate were often susceptible to the other isolates. The highly resistant lines were advanced over several generations by self-pollination. Although these lines were fully homozygous, several lines lost resistance in later generations, indicating that the resistance was unstable. Selection for resistance in these unstable lines did not prevent the occurrence of susceptible progeny in subsequent generations. Therefore, testing over several generations is required to determine the stability of resistance when breeding crops with transgenic virus resistance.     

Biolistic transformation of chrysanthemum with the nucleocapsid gene of tomato spotted wilt virus

In vitro regeneration and biolistic transformation procedures were developed for several commercial chrysanthemum Dendranthema grandiflora Tzvelev, syn. Chrysanthemum morifolium Ramat. cultivars using leaf and stem explants. Studies on the effect of several growth regulators and kanamycin on chrysanthemum regeneration were conducted, and a step-wise procedure to optimize kanamycin selection and recovery of transgenic plants was developed. A population of putative transformed chrysanthemum plants cvs. Blush, Dark Bronze Charm, Iridon, and Tara, was obtained after bombardment with tungsten microprojectiles coated with the binary plasmid pBIN19 containing the nucleocapsid (N) gene of tomato spotted wilt virus (TSWV) and the marker gene neomycin phosphotransferase (NPT II). PCR analysis of 82 putative transgenic plants selected on kanamycin indicated that the majority of the lines (89%) were transformed and contained both genes (71%). However, some transgenic lines contained only one of the genes: either the NPT II (15%) or the TSWV (N) gene (14%). Southern blot analysis on selected transgenic lines confirmed the integration of the TSWV (N) gene into the chrysanthemum genome. These results demonstrate the development of an efficient procedure to transfer genetic material into the chrysanthemum genome and selectively regenerate transgenic chrysanthemum plants at frequencies higher than previously reported.     

Resistance to a DNA and a RNA virus in transgenic plants by using a single chimeric transgene construct

Tomato leaf curl Taiwan virus (ToLCTWV) and Tomato spotted wilt virus (TSWV) are two major tomato viruses that cause serious economic losses. In this study, a partial C2 gene from ToLCTWV and the middle half of the N gene of TSWV were fused as a chimeric transgene to develop multiple virus resistance in transgenic plants. This construct was introduced into Nicotiana benthamiana and tomato by Agrobacterium-mediated transformation. Several transgenic lines showed no symptom post agro-inoculation with ToLCTWV and displayed high resistance to TSWV. The detection of siRNAs indicated that the resistance was via RNA silencing. This study demonstrated that linkage of gene segments from two viruses with distinct genomic organization, one DNA and the other RNA, can confer multiple virus resistance in transgenic plants via gene silencing.     

Combining transgenic and natural resistance to obtain broad resistance to tospovirus infection in tomato (Lycopersicon esculentum mill)

This study was undertaken to develop tomato plants with broad resistanceto tospoviruses which are a major limiting factor to tomato productionworldwide. A nontransgenic tomato line Stevens-Rodale (S-R), six transgenictomato lines expressing the nucleocapsid (N) protein gene of the lettuceisolate of tomato spotted wilt virus (TSWV-BL), and progeny of the crosses between S-Rand three of the transgenic lines homozygous for the N gene were evaluated fortheir resistance to tospovirus infection in greenhouse inoculation tests. S-Rhas the Sw-5 gene that confers resistance to several TSWVisolates. The six transgenic lines showed high levels of resistance wheninoculated with either TSWV-BL or a tomato isolate from Hawaii (TSWV-H).However, these same plants were highly susceptible to the Brazilian isolate ofgroundnut ringspot virus (GRSV-BR). Plants with the Sw-5gene were resistant to TSWV-BL and GRSV-BR, but were susceptible to TSWV-H.When inoculated with any of the three viruses, the F₁ progeny of thecrosses exhibited a susceptible, tolerant, or resistant phenotype with a higherproportion of the plants being either tolerant or resistant. When F₂progeny from F₁ resistant plants of each cross were inoculated withany of the three viruses, a higher proportion of tolerant and resistant plantswas observed compared to the F₁ progeny. Our results show thepotential to obtain broad resistance to tospoviruses by combining transgenicand natural resistance in a single plant.     

Recovery of transgenic peanut (Arachis hypogaea L.) plants from elite cultivars utilizing ACCELL® technology

Transgenic plants of Florunner and Florigiant, two of the most widely cultivated peanut cultivars in the USA, have been developed using the ACCELL® gene delivery method. Shoot meristems of mature embryonic axes were bombarded with gold beads coated with DNA encoding β-glucuronidase (gus), phosphinothricin acetyl transferase (bar), and tomato spotted wilt virus-nucleocapsid protein (tswv-np) genes. Transgenic shoots were identified by screening for GUS activity, and independent transformants were recovered from both cultivars. Molecular analysis of two of these transformants in R₀ and R₁ generations demonstrated the stable integration of the foreign genes into the plant genome. One transgenic plant had one to two copies of the genes integrated into the genome of its progeny, whereas the other had multiple copies. Gus and bar genes exhibited predictable segregation ratios in the R₁ and R₂ generations and were genetically linked. Integration of the bar gene conferred resistance to BASTA^TM, a wide-spectrum herbicide, applied at 500 p.p.m. of active ingredient. Resistance of the transgenic plants to tomato spotted wilt virus is currently being tested under greenhouse conditions. The ACCELL® particle bombardment system is expected to be effective for transformation of a wide variety of commercial peanut cultivars.     

Different effects on ACC oxidase gene silencing triggered by RNA interference in transgenic tomato

RNA interference (RNAi) is a potent trigger for specific gene silencing of expression in a number of organisms and is an efficient way of shutting down gene expression. 1-Aminocyclopropane-1-carboxylate (ACC) oxidase catalyzes the oxidation of ACC to ethylene, a plant growth regulator that plays an important role in the tomato ripening process. In this research, to produce double-stranded (ds)RNA of tomato ACC oxidase, we linked the sense and antisense configurations of DNA fragments with 1,002-bp or 7-nt artificially synthesized fragments, respectively, and then placed these under the control of a modified cauliflower mosaic virus 35S promoter. The dsRNA expression unit was successfully introduced into tomato cultivar Hezuo 906 by Agrobacterium tumefaciens-mediated transformation. Molecular analysis of 183 transgenic plants revealed that the dsRNA unit was integrated into the tomato genome. With respect to the construct with the 1,002-bp linker, the severity of phenotypes indicated that 72.3% of the transformed plants had non-RNA interference, about 18.1% had semi-RNA interference, and only 9.6% had full-RNA interference. However when the construct with the 7-nt linker was used for transformation, the results were 13.0%, 18.0%, and 69.0%, respectively, indicating that the short linker was more efficient in RNAi of transgenic tomato plants. When we applied this fast way of shutting down the ACC oxidase gene, transgenic tomato plants were produced that had fruit which released traces of ethylene and had a prolonged shelf life of more than 120 days. The RNA and protein analyses indicated that there was non-RNA interference, semi-RNA interference and full-RNA interference of ACC oxidase in the transgenic tomato plants.     

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.     

The nucleotide sequence of the S RNA of Impatiens necrotic spot virus, a novel tospovirus.

Impatiens necrotic spot virus (INSV) shares a number of properties with tomato spotted wilt virus (TSWV), the type species of the genus tospovirus within the family Bunyaviridae. INSV, however, differs from TSWV in plant host range and serology. In order to define the genomic structure and the taxonomic status of this TSWV-like virus, the nucleotide sequence of its genomic S RNA segment has been determined. The molecular data obtained demonstrate that, like TSWV, INSV has an ambisense S RNA molecule, encoding a non-structural protein in viral sense and the nucleocapsid protein in viral complementary sense. The level of nucleotide sequence homology between their S RNAs, as well as the divergence in amino acid sequence homology of their gene products, confirm previous conclusions from serological studies that INSV and TSWV represent distinct virus species within the newly created genus, tospovirus.     

Agrobacterium-mediated transformation of cultivated tomato with construct carrying the nucleoprotein (N) gene from tomato spotted wilt virus (TSWV)

Significant yield losses in commercial tomato production caused by tomato spotted wilt virus (TSWV) are the reason why we have undertaken studies on resistance to this pathogen. One of the possible sources of resistance can be the incorporation of the nucleoprotein N viral gene by Agrobacterium transformation. The N gene was introduced into three Lycopersicon esculentum forms. Out of the total of 3044 cotyledon explants 14.7% regenerated shoots, but only a few were rooted on medium containing kanamycin. The preliminary analysis indicated that 18 plants are putative transformants.     

Overcoming host- and pathogen-mediated resistance in tomato and tobacco maps to the M RNA of Tomato spotted wilt virus

A viral genetic system was used to map the determinants of the ability of Tomato spotted wilt virus (TSWV) to overcome the R gene (Sw-5) in tomato and the resistance conferred by the nucleocapsid gene of TSWV (N gene) in tobacco. A complete set of reassortant genotypes was generated from TSWV isolates A and D. TSWV-A was able to overcome the Sw-5 gene in tomato and the TSWV N gene in tobacco, whereas TSWV-D was repressed by both forms of resistance. The ability to overcome both forms of resistance was associated with the M RNA segment of TSWV-A (M(A)). Overcoming the Sw-5 gene was linked solely to the presence of M(A), and the ability of M(A) to overcome the TSWV N gene was modified by the L RNA and the S RNA of TSWV-A, which is consistent with previous reports that suggest that the nucleocapsid gene is not the primary determinant for overcoming the nucleocapsid-mediated resistance. Sequence analysis of the M RNA segment of TSWV-A, -D, and the type isolate BR-01 revealed multiple differences in the coding and noncoding regions, which prevented identification of the resistance-breaking nucleotide sequences.     

Peanut yellow spot virus: A distinct tospovirus species based on serology and nucleic acid hybridisation

Nucleocapsids of peanut yellow spot virus (PYSV), purified from peanut (= groundnut) plant tissue, contained a protein with a molecular mass of 29 kDa. In ELISA and immuno-blot analysis the virus did not react with tomato spotted wilt virus (TSWV), Impatiens necrotic spot virus (INSV) and peanut bud necrosis virus (PBNV) antisera. PYSV contained three RNA species, a large (L) RNA (c.8900 nucleotides), a medium (M) RNA (c.4800 nucleotides) and a small (S) RNA (c.3000 nucleotides), similar to other tospoviruses. In addition, a fourth RNA species of approximately 1800 nucleotides was also present in purified preparations. Hybridisation analysis under high stringency conditions revealed no hybridisation between PYSV RNAs and cDNA probes representing the nucleocapsid (N) gene, the glycoprotein (GP) gene and the 3' half of the RNA polymerase gene of PBNV. PYSV genomic RNAs also failed to hybridise with cDNA probes from the GP genes of TSWV and INSV. In reciprocal tests, the cDNA clones of PYSV S and M RNAs did not hybridise with any of the PBNV RNAs. Based on the absence of serological relationships between PYSV and PBNV, TSWV and INSV and lack of nucleotide homology based on hybridisation studies between the PYSV RNAs and cDNA clones from PBNV, TSWV and INSV, PYSV should be considered as a distinct species of the genus Tospovirus under a new serogroup, putatively designated ‘V’.     

Grafting on a Non-Transgenic Tolerant Tomato Variety Confers Resistance to the Infection of a Sw5-Breaking Strain of Tomato spotted wilt virus via RNA Silencing

RNA silencing controls endogenous gene expression and drives defensive reactions against invasive nucleic acids like viruses. In plants, it has been demonstrated that RNA silencing can be transmitted through grafting between scions and silenced rootstocks to attenuate virus and viroid accumulation in the scions. This has been obtained mostly using transgenic plants, which may be a drawback in current agriculture. In the present study, we examined the dynamics of infection of a resistance-breaking strain of Tomato spotted wilt virus (RB-TSWV) through the graft between an old Apulian (southern Italy) tomato variety, denoted Sl-Ma, used as a rootstock and commercial tomato varieties used as scions. In tests with non-grafted plants, Sl-Ma showed resistance to the RB-TSWV infection as viral RNA accumulated at low levels and plants recovered from disease symptoms by 21 days post inoculation. The resistance trait was transmitted to the otherwise highly susceptible tomato genotypes grafted onto Sl-Ma. The results from the analysis of small RNAs hallmark genes involved in RNA silencing and virus-induced gene silencing suggest that RNA silencing is involved in the resistance showed by Sl-Ma against RB-TSWV and in scions grafted on this rootstock. The results from self-grafted susceptible tomato varieties suggest also that RNA silencing is enhanced by the graft itself. We can foresee interesting practical implications of the approach described in this paper.     

兔防御素NP-1基因在转基因番茄中表达的初步研究

兔防御素ＮＰ－１是α－防御素的一种,含３３个氨基酸残基。最初从兔子的多形核嗜中性细胞中分离出来。它对革兰氏阴性菌、革兰氏阳性菌、分枝杆菌、真菌、被膜病毒以及ＨＩＶ病毒都有不同程度的抑制作用。兔防御素ＮＰ－１所带阳离子较多,可抗不具代谢活性的靶细胞。实验中将兔防御素ＮＰ－１基因构建到植物表达载体中,通过根瘤农杆菌介导转入番茄,得到了转基因番茄植株。对转基因番茄植株进行了ＰＣＲ、Ｓｏｕｔｈｅｍ杂交、Ｎ     

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.     

Differential Susceptibilities between Leaf Disks and Plants in the Transmission of Tomato Spotted Wilt Virus by Frankliniella occidentalis to TSWV Hosts and Transgenic Plants

The efficiency by which tomato spotted wilt virus (TSWV) was transmitted to plants and leaf disks by the vector Frankliniella occidentalis , was analysed. The virus was efficiently transmitted to Datura stramonium, Impatiens sp. and tobacco plants, i.e. 60–100% of the plants became infected when 1–3 viruliferous thrips were confined per plant for a period of 3 days. However, lettuceexhibited a lower susceptibility since only 25% of the test plants were infected when challenged by 10 viruliferous thrips per plant for 3 days. In contrast, complete resistance was found when transgenic tobacco plants, expressing the nucleocapsid protein of TSWV, were challenged with up to 10 viruliferous thrips per plant, whereas all non-transgenic control plants were infected when 5 viruliferous thrips per plant were used. To improve and accelerate the tramission studies, the applicability of leaf disks in these studies was tested. Leaf disks of 16 different plant species appeared to be highly susceptible. Infection ratings ranging from 51.6 to 95.0% were obtained when one viruliferous adult was placed singly on these leaf disks for a period for 24 h. The leaf disk assay was also employed to screen resistance of transgenic plants expressing the nucleocapsid protein of TSWV. One transgenic tomato line displayed complete immunity whereas a second line appeared to be susceptible. For the transgenic tobacco line, positive ELISA reactions were found for a few leaf disks (7.5%) suggesting that some virus replication did occur. However, the ELISA readings for these disks were significantly lower than those for leaf disks of non-transgenic controls. Finally, the significance of the use of the leaf disks and test plants in virus-vector studies is discussed.