Traditional and transgenic strategies for controlling tomato-infecting begomoviruses (original) (raw)
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Breeding for resistance to begomovirus in tropic-adapted tomato genotypes
Plant Breeding, 2004
Four tomato lines introgressed from Lycopersicon chilense were compared with the commercial F1 hybrids ‘ARO 8479’ and ‘HA 3108’, which are tolerant to Tomato yellow leaf curl virus, and the cv. ‘Campbell 28’ as a susceptible control. Resistance was evaluated by the use of grafted diseased scions as well as in a field trial where plants infected by viruliferous whiteflies and disease-free plants were transplanted in paired rows. The new lines LD 3, LD 4, LD 5 and LD 6 showed no disease symptoms after grafting or in the field trial. Virus accumulation at 60 days after transplanting was low in the infected plants: 0.09, 0.60, 1.00 and 0.50 ng, respectively. No fruit-set or yield losses were registered under the high temperature conditions prevalent in the trial, in which lines LD 5 and LD 6 were better adapted to tropical conditions. Viral DNA concentrations were over 1000 ng in the cvs.‘Campbell 28′,‘ARO 8479’ and ‘HA 3108’. The last two are considered tolerant as they were asymptomatic or had mild symptoms, respectively, but achieved acceptable yields in the trial. By contrast, virus had a negative effect on fruit-set, number of fruit per plant and total yield in the cv.‘Campbell 28’.
Virus Research, 2009
Two tomato geminivirus species co-exist in protected crops in Sicily, Tomato yellow leaf curl Sardinia virus (TYLCSV, found in 1989) and Tomato yellow leaf curl virus (TYLCV, found in 2002), and mixed infections have been detected. In a field survey conducted in 2004, the viral intergenic region (IR) was amplified from infected plants, and molecules apparently hybrid between the two species were found, but only in plants where one or both parental species were also present. Two of these hybrids, named 2/2 and 2/5, were isolated and infectious clones were obtained. They were both readily whitefly-transmitted to tomato plants; clone 2/5 produced symptoms typical of TYLCSV and TYLCV, while clone 2/2 produced more severe symptoms, with leaves showing downward curling and rugosity. Sequence analysis showed that both 2/2 and 2/5 are newly generated hybrids, with two recombination sites each. One site, common to both hybrids, is in the stem-loop of the IR. The other is close to the 3 -end of the CP ORF in 2/5 and within the Rep ORF in 2/2. Thus, the 2/2 hybrid virus has a hybrid Rep protein, with the 202 amino-terminal aa from TYLCV and the remaining 155 aa from TYLCSV. Replication assays in leaf disc indicated a lower replicative capacity with respect to parental viruses, a fact that might help to explain why plants infected only by a recombinant have not been found so far.
Acta horticulturae
Tomato yellow leaf curl virus (TYLCV), a whitefly transmitted geminivirus, is the major limiting factor for tomato production in many countries in the Middle East, Europe, Africa and was reported in North and South America. Development of resistant varieties represents the most economical management measure. DNA sequences of the six major strains of TYLCV reported in the Middle East and Europe were aligned and sequences with more than 21 conserved nucleotides were determined. Three primer pairs were designed and used in PCR assays to amplify three overlapping regions of 240, 355 and 505bps. The amplicons were cloned in pHELLSGATE 2 using BP and LR clonase. This protocol allows the insertion, in one step, of the genes of interest in sense and antisense orientation with an intron in between. Once introduced into the plant it will be transcribed into dsRNA leading to post-transcriptional gene silencing of homologous genes. Colonies of Agrobacterium strain LBA 4404 were transformed with pHELLSGATE vectors carrying each of the three constructs and used to inoculate cotyledons of the tomato variety MP1. The produced transgenic tomato seedlings with the 505 bp construct developed TYLCV like symptoms upon inoculation but recovered rapidly thereafter. However, TYLCV concentration in the recovered tissue, as determined by semi-quantitative PCR, does not seem to have been reduced considerably. This construct includes partial pV1, pC2, and pC3, indicating that part of this region may play a significant role in development of TYLCV symptoms.
Since 1991, farmers in south and central Jamaica have reported losses of up to 100% in their tomato and pepper crops. Increased losses were associated with viral diseases transmitted by the whitefly Bemisia tabaci. Owing to the potential threat to tomato and pepper production, the overall aim of this project was to provide an increased technological base for the development of disease management strategies for whitefly-transmitted geminiviral diseases affecting pepper and tomato in Jamaica. The specific objectives were: 1) To molecularly and biologically characterize the whitefly-transmitted geminiviruses infecting vegetable crops including tomatoes, peppers, and weeds in Jamaica. 2) To develop rapid diagnostic systems for the identification of geminiviruses associated with tomatoes and peppers, and use these techniques to provide geminivirus diagnosis for the Caribbean Basin countries. 3) To screen cultivated and wild species of tomato genotypes for resistance/tolerance to tomato-infecting geminiviruses. 4) To evaluate antiviral strategies for use in engineering tomatoes with resistance to infections of tomato yellow leaf curl geminivirus. 5) To produce and select begomovirus-resistant tomato varieties suitable for the Jamaica market. Geminivirus DNA probes, polymerase chain reaction and nucleotide sequencing were used to identifjr and characterize geminiviruses fiom Jamaica and Barbados. The monopartite Tomato yellow leaf curl virus (TYLCV, Israeli strain), was identified in Lycopersicon esculentum (tomato) in Jamaica. Another, new, bipartite geminivirus, Tomato dwarf leaf curl virus (TDLCV) was found in tomato and Scotch Bonnet pepper (Capsicum chinense). Two distinct viruses infecting cabbage were identified. The common weeds Sida sp and Wissadula amplissima were found to contain geminiviruses Sida golden mosaic virus (SiGMV-JM) and Wissadula golden mosaic virus (WGMV), respectively. Three distinct geminiviruses were found in the weed Macroptilium lathyroides: SiGMV-JM and Macroptilium golden mosaic virus strains 1 and 2 (MacGMV-JM1, MacGMV-JM2). In Barbados, tomato, muskmelon and watermelon contained the same bipartite gerninivirus, whereas, two distinct geminiviruses were associated with M. lathyroides. Phylogenetic relationships of the Jamaican viruses with other Western Hemisphere geminiviruses indicate that crop-and weed-infecting geminiviruses fiom Jamaica and Barbados are distinct, highly diverse, and have several geographical origins. It can be concluded that weeds are not host to crop-infecting geminiviruses in Jamaica. A new strategy to genetically engineering tomato for resistance to TYLCV was developed. The rep mutant developed by this project in collaboration with the University of Wisconsin is now being engineered into tomato in the Jamaican laboratory. The virus coat protein gene V1 and the Rep protein gene rep have been used to transform tomato plants in Israel and Jamaica. A transgenic tomato line expressing the coat protein of TYLCV have been shown to be immune to whitefly-mediated inoculation at the University of Jerusalem. This line has been crossed with commercial varieties and hybrids are tested. Tomato lines have been developed by the Hebrew University of Jerusalem using the wild tomato species L. hirsutum as a source for resistance to TYLCV. These lines showed high levels of resistance in field tests and in the laboratory. Hybrids have been made and a largescale field trial has been conducted in Jamaica. Some of the lines showed excellent levels of resistance and marketable h i t qualities. Cooperation between the project partners has been implemented by several long-and shortterm research visits of the researchers and their collaborators in the participating laboratories. '~idavski, F., 2~c~l a s h a n , D., 3~c~a u g h l i n
Phytopathology, 2004
Use of Tomato yellow leaf curl virus (TYLCV) Rep gene sequences to engineer TYLCV resistance in tomato. Phytopathology 94:490-496. Tomato yellow leaf curl virus (TYLCV), a member of the genus Begomovirus (family Geminiviridae), causes severe losses in tomato production in the tropics and subtropics. In order to generate engineered resistance, eight different constructs of the TYLCV replication-associated protein (Rep) and C4 gene sequences were tested in transformed tomato inbred lines. Transgenic plants were screened for resistance to TYLCV using viruliferous whiteflies. No symptoms were observed and no TYLCV genomic DNA was detected by both hybridization and polymer-ase chain reaction in progenies of plants transformed with three constructs. This resistance was observed in plants that contained one of the following transgenes: 2/5Rep (81 nucleotides [nt] of the intergenic region [IR] plus 426 nt of the 5′ end of the TYLCV Rep gene), ∆2/5Rep (85 nt of the IR plus 595 nt of the 5′ end of the TYLCV Rep gene in the antisense orientation), and Rep∆2/5Rep (81 nt of the IR, the entire Rep gene, and 41 nt 3′ to the end of the Rep gene fused to ∆2/5Rep). Our study differs from other transgenic Geminivirus resistance reports involving the Rep gene in that viruliferous whiteflies were used for challenge inoculation instead of agroinoculation or biolistic inoculation, and TYLCV resistance was evaluated under field conditions.
Lanai: A small, fast growing tomato variety is an excellent model system for studying geminiviruses
Journal of Virological Methods, 2018
Geminiviruses are devastating single-stranded DNA viruses that infect a wide variety of crops in tropical and subtropical areas of the world. Tomato, which is a host for more than 100 geminiviruses, is one of the most affected crops. Developing plant models to study geminivirus-host interaction is important for the design of virus management strategies. In this study, "Florida Lanai" tomato was broadly characterized using three begomoviruses (Tomato yellow leaf curl virus, TYLCV; Tomato mottle virus, ToMoV; Tomato golden mosaic virus, TGMV) and a curtovirus (Beet curly top virus, BCTV). Infection rates of 100% were achieved by agroinoculation of TYLCV, ToMoV or BCTV. Mechanical inoculation of ToMoV or TGMV using a microsprayer as well as whitefly transmission of TYLCV or ToMoV also resulted in 100% infection frequencies. Symptoms appeared as early as four days post inoculation when agroinoculation or bombardment was used. Symptoms were distinct for each virus and a range of features, including plant height, flower number, fruit number, fruit weight and ploidy, was characterized. Due to its small size, rapid growth, ease of characterization and maintenance, and distinct responses to different geminiviruses, "Florida Lanai" is an excellent choice for comparing geminivirus infection in a common host.
Advances and Prospects of Virus-Resistant Breeding in Tomatoes
International Journal of Molecular Sciences
Plant viruses are the main pathogens which cause significant quality and yield losses in tomato crops. The important viruses that infect tomatoes worldwide belong to five genera: Begomovirus, Orthotospovirus, Tobamovirus, Potyvirus, and Crinivirus. Tomato resistance genes against viruses, including Ty gene resistance against begomoviruses, Sw gene resistance against orthotospoviruses, Tm gene resistance against tobamoviruses, and Pot 1 gene resistance against potyviruses, have been identified from wild germplasm and introduced into cultivated cultivars via hybrid breeding. However, these resistance genes mainly exhibit qualitative resistance mediated by single genes, which cannot protect against virus mutations, recombination, mixed-infection, or emerging viruses, thus posing a great challenge to tomato antiviral breeding. Based on the epidemic characteristics of tomato viruses, we propose that future studies on tomato virus resistance breeding should focus on rapidly, safely, and e...
Begomovirus Disease: The most devastating disease of cultivated tomato
2013
Recently, several begomoviruses (genus Begomovirus; family Geminiviridae) have been associated with leaf curl and yellow leaf curl symptoms in tomato crops worldwide. These viruses are transmitted by the whitefly Bemisia tabaci (Genn.) sibling species group, and they have a genome of single stranded (ss) DNA (about 2.7-2.8 kb) with an organization typical for Old World monopartite begomoviruses. In addition, many monopartite begomoviruses are associated with alphasatellite and betasatellite molecules that may influence symptom development, pathogenicity, and virulence. Wild, uncultivated plant species are commonly infected with viruses, and may serve as reservoirs of viruses also pathogenic to cultivated plants, and can exert dynamic forces on the ecology of natural systems.
Phytopathology, 2004
Use of Tomato yellow leaf curl virus (TYLCV) Rep gene sequences to engineer TYLCV resistance in tomato. Phytopathology 94:490-496. Tomato yellow leaf curl virus (TYLCV), a member of the genus Begomovirus (family Geminiviridae), causes severe losses in tomato production in the tropics and subtropics. In order to generate engineered resistance, eight different constructs of the TYLCV replication-associated protein (Rep) and C4 gene sequences were tested in transformed tomato inbred lines. Transgenic plants were screened for resistance to TYLCV using viruliferous whiteflies. No symptoms were observed and no TYLCV genomic DNA was detected by both hybridization and polymer-ase chain reaction in progenies of plants transformed with three constructs. This resistance was observed in plants that contained one of the following transgenes: 2/5Rep (81 nucleotides [nt] of the intergenic region [IR] plus 426 nt of the 5′ end of the TYLCV Rep gene), ∆2/5Rep (85 nt of the IR plus 595 nt of the 5′ end of the TYLCV Rep gene in the antisense orientation), and Rep∆2/5Rep (81 nt of the IR, the entire Rep gene, and 41 nt 3′ to the end of the Rep gene fused to ∆2/5Rep). Our study differs from other transgenic Geminivirus resistance reports involving the Rep gene in that viruliferous whiteflies were used for challenge inoculation instead of agroinoculation or biolistic inoculation, and TYLCV resistance was evaluated under field conditions.
Viruses, 2015
The tomato yellow leaf curl disease (TYLCD) causes severe damage to tomato (Solanum lycopersicum L.) crops throughout tropical and subtropical regions of the world. TYLCD is associated with a complex of single-stranded circular DNA plant viruses of the genus Begomovirus (family Geminiviridae) transmitted by the whitefy Bemisia tabaci Gennadius (Hemiptera: Aleyrodidae). The tomato inbred line TX 468-RG is a source of monogenic recessive resistance to begomoviruses derived from the hybrid cv. Tyking F 1. A detailed analysis of this germplasm source against tomato yellow leaf curl virus-Israel (TYLCV-IL), a widespread TYLCD-associated virus, showed a significant restriction to systemic virus accumulation even under continuous virus supply. The resistance was effective in limiting the onset of TYLCV-IL in tomato, as significantly lower primary spread of the virus occurred in resistant plants. Also, even if a limited number of resistant Viruses 2015, 7 2519 plants could result infected, they were less efficient virus sources for secondary spread owing to the impaired TYLCV-IL accumulation. Therefore, the incorporation of this resistance into breeding programs might help TYLCD management by drastically limiting TYLCV-IL spread.