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.     

Variety,mulch and stage of inoculation effects on incidence of tomato spotted wilt virus disease in cucumber (Cucumis sativus L.)

Abstract

Tomato spotted wilt virus (TSWV) vectored by thrips is one of the major diseases affecting cucumber yield. Control of thrips is an underlying factor in its management. A study was conducted to determine the effect of time of inoculation, variety and mulch on disease incidence. Four varieties were inoculated with TSWV at cotyledon, 3 – 4 leaf and flower bud stages in a RCBD experiment replicated four times in a greenhouse. In the field, a 2×8 factorial design where two cucumber varieties were raised on seven types of mulches (red, yellow, silver, clear, black, white, and straw) with unmulched plots as controls was used. Variety Marketer was more tolerant to the disease compared to other varieties. Most varieties were generally tolerant to TSWV at cotyledonous but susceptible at 3 – 4 leaf and flower bud stages. Silver and clear mulches significantly suppressed thrip populations, yield and quality under field conditions.     

Developmentally regulated Arabidopsis thaliana susceptibility to tomato spotted wilt virus infection

Tomato spotted wilt virus (TSWV) is one of the most devastating plant viruses and often causes severe crop losses worldwide. Generally, mature plants become more resistant to pathogens, known as adult plant resistance. In this study, we demonstrated a new phenomenon involving developmentally regulated susceptibility of Arabidopsis thaliana to TSWV. We found that Arabidopsis plants become more susceptible to TSWV as plants mature. Most young 3-week-old Arabidopsis were not infected by TSWV. Infection of TSWV in 4-, 5-, and 6-week-old Arabidopsis increased from 9%, 21%, and 25%, respectively, to 100% in 7- to 8-week-old Arabidopsis plants. Different isolates of TSWV and different tospoviruses show a low rate of infection in young Arabidopsis but a high rate in mature plants. When Arabidopsis dcl2/3/4 or rdr1/2/6 mutant plants were inoculated with TSWV, similar results as observed for the wild-type Arabidopsis plants were obtained. A cell-to-cell movement assay showed that the intercellular movement efficiency of TSWV NSm:GFP fusion was significantly higher in 8-week-old Arabidopsis leaves compared with 4-week-old Arabidopsis leaves. Moreover, the expression levels of pectin methylesterase and β-1,3-glucanase, which play critical roles in macromolecule cell-to-cell trafficking, were significantly up-regulated in 8-week-old Arabidopsis leaves compared with 4-week-old Arabidopsis leaves during TSWV infection. To date, this mature plant susceptibility to pathogen infections has rarely been investigated. Thus, the findings presented here should advance our knowledge on the developmentally regulated mature host susceptibility to plant virus infection.     

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     

Mapping the Sw-5 locus for tomato spotted wilt virus resistance in tomatoes using RAPD and RFLP analyses

The Sw-5 locus confers dominant resistance to tomato spotted wilt virus (TSWV). To map the location and facilitate the identification of markers linked to Sw-5 we developed a pair of near-isogenic lines (NILs) and an F₂ Lycopersicon esculentum x L. pennellii population segregating for resistance to TSWV. DNA from the NILs was analyzed using 748 random 10-mer oligonucleotides to discern linked molecular markers using a random amplified polymorphic DNA (RAPD) approach. One random primer (GAGCACGGGA) was found to produce a RAPD band of about 2200 bp that demonstrates linkage to Sw-5. Data from co-segregation of resistance and restriction fragment length polymorphisms (RFLPs) in a F₂ interspecific population position Sw-5 between the markers CT71 and CT220 near the telomere of the long arm of chromosome 9.     

Screening Capsicum germplasm for resistance to tomato spotted wilt virus (TSWV)

Resistance conferred by the Tsw locus from Capsicum chinense against Tomato spotted wilt virus (TSWV) has been widely used in breeding programmes. Nevertheless, this resistance depends on inoculation conditions, and isolates able to overcome it have already been detected. In this work 29 accessions of several Capsicum species have been mechanically inoculated with TSWV to identify new sources of resistance. Five accessions showed variable percentages of resistant plants, two of which did not show local lesions on inoculated leaves, suggesting that the response was not mediated through hypersensitivity. Two of these accessions also had a remarkable reduced viral accumulation compared to susceptible control. ECU‐973., a C. chinense accession, showed the best performance against TSWV, with 100% resistant plants. This response was confirmed after mechanical inoculation with three different TSWV isolates. The resistance was maintained when the accession was inoculated with TSWV using a high pressure of viruliferous thrips. These results open new possibilities in the development of a durable resistance to TSWV in pepper.     

Variation in thrips species composition in field crops and implications for tomato spotted wilt epidemiology in North Carolina

Thrips were surveyed in tomato spotted wilt-susceptible crops in five areas across North Carolina. Tomato, pepper, and tobacco plants in commercial fields were sampled and 30 species of thrips were collected over a 3-year period. The most common species overall was Frankliniella tritici (Fitch). The most common thrips species that are known to vector Tomato Spotted Wilt Virus (TSWV) were F. fusca (Hinds), and F. occidentalis (Pergande). Relatively low numbers of Thrips tabaci Lindeman, another reported vector, were collected. The spatial and temporal occurrence of vectors varied with sampling method, crop species, region of North Carolina, and localized areas within each region. In a laboratory experiment, no difference was detected between the ability of F. fusca and F. occidentalis to acquire and transmit a local isolate of TSWV. Based on vector efficiency and occurrence, F. fusca is considered the most important vector of TSWV in tobacco, whereas both F. fusca and F. occidentalis are important vectors of TSWV in tomato and pepper.     

Monoclonal Antibodies against the Recombinant Nucleocapsid Protein of Tomato spotted wilt virus and its Application in Virus Detection

Tomato spotted wilt virus (TSWV) is the type member of the tospovirus genus and causes significant losses in a wide range of economically important ornamental and vegetable crops worldwide. The nucleocapsid gene, located on the ambisense S RNA segment of TSWV was expressed in Escherichia coli using pET-32a as vector and correct expression of recombinant protein was confirmed by Western blot using an anti-TSWV monoclonal antibody (MAb). The recombinant protein was purified using Ni-NTA agarose and the purified protein was used for the production of MAbs. Three murine MAbs against the recombinant nucleocapsid protein were produced. Triple antibody sandwich enzyme-linked immunosorbent assay and immunocapture RT-PCR methods were then established for reliable and efficient detection of TSWV using the produced MAbs.     

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.     

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.     

Fungicidal and Cytotoxic Activity of a Capsicum chinense Defensin Expressed by Endothelial Cells

Plant defensins are antimicrobial peptides that exhibit mainly antifungal activity against a broad range of plant fungal pathogens. However, their actions against Candida albicans have not been extensively studied. The mRNA for γ-thionin, a defensin from Capsicum chinense, has been expressed in bovine endothelial cells. The conditioned medium of these cells showed antifungal activity on germ tube formation (60–70% of inhibition) and on the viability of C. albicans (70–80% of inhibition). Additionally, C. albicans was not able to penetrate transfected cells. Conditioned medium from these cells also inhibited the viability (80%) of the human tumor cell line, HeLa.     

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     

Temporal and spatial dynamics of Tomato spotted wilt virus and its vector in a potato crop in Argentina

The nature of spatial and temporal dynamics of Tomato spotted wilt virus (TSWV) and its vector in a potato crop cv. Innovator without insecticide application is analysed. Seed tuber was analysed for the presence of TSWV as a source of initial inoculum. The presence of plants with symptoms of TSWV was evaluated by visual observation and DAS‐ELISA analysis to confirm the virus infection. Thrips species were collected from leaves and inflorescences and identified under stereomicroscope. The distribution of symptomatic plants and thrips species was recorded five times at 14 days intervals. The initial seed tuber infection was of 1.1%. Disease incidence was 0% at 29 days after planting (DAP), 0.2% at 43 DAP, 2.2% at 56 DAP, 11.6% at 70 DAP and 14.6% at 84 DAP. The progress of the disease was adequately described by a Logistic model [y = 0.15/(1 + 1205372.93 × exp (?0.22 × DAP))]. Thrips vector species identified as resident in the crop during the whole cycle were Thrips tabaci (n = 423), Frankliniella occidentalis (n = 141) and as occasional species, F. schultzei (n = 34) and F. gemina (n = 5) were found. At 43 and 56 DAP a random distribution pattern was observed and the thrips species found were T. tabaci (n = 188) and F. occidentalis (n = 105). An aggregated pattern was determined at 70 and 84 DAP. Spatial patterns of the disease spread suggest a polycyclic epidemic with TSWV secondary spread in the potato crop. Multiple control measures were deduced from these epidemiological results like virus testing in tubers, removal of external virus infection sources and thrips control.     

Mapping strategy for resistance genes in tomato based on RFLPs between cultivars: Cf9 (resistance to Cladosporium fulvum) on chromosome 1

Summary The contribution of introgressed regions derived from wild species to the genetic variation within the species of Lycopersicon esculentum was investigated by comparing the RFLP patterns of 2 introgression-free, obsolete cultivars (Moneymaker and Premier) and a modern cultivar (Sonatine) that carries at least 5 introgressed resistance genes. In this analysis 195 mapped nuclear markers were used in combination with 6 restriction enzymes. Among the 1170 probe-enzyme combinations tested, only 3 showed a polymorphism between the 2 introgression-free cultivars. On the other hand 24 probe-enzyme combinations were found to exhibit polymorphisms between Moneymaker and Sonatine. These represented ten polymorphic loci distributed among 5 linkage groups on chromosomes 1, 3, 4, 6, and 9.On the assumption that most of the polymorphic loci corresponded to introgressed chromosome segments of wild species carrying resistance genes, linkages between these loci and the component resistance genes were examined by RFLP analysis of pairs of near-isogenic lines differing only for one particular resistance gene, and a variety of commercial cultivars having different resistance gene compositions. Two of the polymorphic linkage groups could thus be ascribed to resistance genes whose map positions were already known: Cf2 on chromosome 6 and Tm2a on chromosome 9, whereas another marker, TG301 on chromosome 1, could be assigned to the Cladosporium fulvum resistance gene Cf9 with a hitherto disputable map position. By linkage analysis of a segregating F₂ population the genetic distance between the Cf9 gene and the marker TG301 was estimated at 5.5 ± 2.3 cM.     

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.     

Identification of two AFLP markers linked to bacterial wilt resistance in tomato and conversion to SCAR markers

Tomato bacterial wilt (BW) incited by Ralstonia solanacearum is a constraint on tomato production in tropical, subtropical and humid regions of the world. In this paper, we present the results of a research aimed at the identification of PCR-based markers amplified fragment length polymorphism (AFLP) linked to the genes that confer resistance to tomato BW. To this purpose, bulked segregant analysis was applied to an F₂ population segregating for the BW resistant gene and derived from the pair-cross between a BW resistant cultivar T51A and the susceptible cultivar T9230. Genetic analysis indicated that tomato BW was conferred by two incomplete dominant genes. A CTAB method for total DNA extraction, developed by Murray and Thompson with some modifications was used to isolation the infected tomato leaves. Thirteen differential fragments were detected using 256 primer combinations, and two AFLP markers were linked to the BW resistance. Subsequently, the AFLP markers were converted to co-dominant SCAR markers, named TSCAR_AAT/CGA and TSCAR_AAG/CAT. Linkage analysis showed that the two markers are on the contralateral side of TRSR-1. Genetic distance between TSCAR_AAT/CGA and TRS-1 was estimated to 4.6 cM, while 8.4 cM between TSCAR_AAG/CAT and TRS-1.     

Molecular mapping of wilt resistance genes in chickpea

Fusarium wilt is a widespread and serious chickpea disease caused by the soil-borne fungus Fusarium oxysporum f.sp. ciceri (Foc). We evaluated an F₉ recombinant inbred line population of chickpea for resistance to three Foc races (1, 2 and 3) in pot culture experiments and identified flanking and tightly linked DNA markers for the resistance genes. The simple sequence repeat markers H3A12 and TA110 flanked the Foc1 locus at 3.9 and 2.1 cM, respectively, while Foc2 was mapped 0.2 cM from TA96 and 2.7 cM from H3A12. The H1B06y and TA194 markers flanked the Foc3 locus at 0.2 and 0.7 cM, respectively. These markers were also validated using 16 diverse chickpea genotypes. Identification of tightly linked flanking markers for wilt resistance genes will be useful for their exploitation in breeding programs and to understand the mechanism of resistance and evolution of the genes. S. J. M. Gowda and P. Radhika contributed equally to this study.