Tomato Yellow Leaf Curl Virus Research Articles

Emerging evidence of seed transmission of begomoviruses: implications in global circulation and disease outbreak

Begomoviruses (family Geminiviridae) are known for causing devastating diseases in fruit, fibre, pulse, and vegetable crops throughout the world. Begomoviruses are transmitted in the field exclusively through insect vector whitefly (Bemisia tabaci), and the frequent outbreaks of begomoviruses are attributed largely due to the abundance of whitefly in the agri-ecosystem. Begomoviruses being phloem-borne were known not be transmitted through seeds of the infected plants. The recent findings of seed transmission of begomoviruses brought out a new dimension of begomovirus perpetuation and dissemination. The first convincing evidence of seed transmission of begomoviruses was known in 2015 for sweet potato leaf curl virus followed by several begomoviruses, like bhendi yellow vein mosaic virus, bitter gourd yellow mosaic virus, dolichos yellow mosaic virus, mungbean yellow mosaic virus, mungbean yellow mosaic India virus, pepper yellow leaf curl Indonesia virus, tomato leaf curl New Delhi virus, tomato yellow leaf curl virus, tomato yellow leaf curl Sardinia virus, and okra yellow mosaic Mexico virus. These studies brought out two perspectives of seed-borne nature of begomoviruses: (i) the presence of begomovirus in the seed tissues derived from the infected plants but no expression of disease symptoms in the progeny seedlings and (ii) the seed infection successfully transmitted the virus to cause disease to the progeny seedlings. It seems that the seed transmission of begomovirus is a feature of a specific combination of host-genotype and virus strain, rather than a universal phenomenon. This review comprehensively describes the seed transmitted begomoviruses reported in the last 9 years and the possible mechanism of seed transmission. An emphasis is placed on the experimental results that proved the seed transmission of various begomoviruses, factors affecting seed transmission and impact of begomovirus seed transmission on virus circulation, outbreak of the disease, and management strategies.

Gibberellin Positively Regulates Tomato Resistance to Tomato Yellow Leaf Curl Virus (TYLCV).

Tomato yellow leaf curl virus (TYLCV) is a prominent viral pathogen that adversely affects tomato plants. Effective strategies for mitigating the impact of TYLCV include isolating tomato plants from the whitefly, which is the vector of the virus, and utilizing transgenic lines that are resistant to the virus. In our preliminary investigations, we observed that the use of growth retardants increased the rate of TYLCV infection and intensified the damage to the tomato plants, suggesting a potential involvement of gibberellic acid (GA) in the conferring of resistance to TYLCV. In this study, we employed an infectious clone of TYLCV to inoculate tomato plants, which resulted in leaf curling and growth inhibition. Remarkably, this inoculation also led to the accumulation of GA3 and several other phytohormones. Subsequent treatment with GA3 effectively alleviated the TYLCV-induced leaf curling and growth inhibition, reduced TYLCV abundance in the leaves, enhanced the activity of antioxidant enzymes, and lowered the reactive oxygen species (ROS) levels in the leaves. Conversely, the treatment with PP333 exacerbated TYLCV-induced leaf curling and growth suppression, increased TYLCV abundance, decreased antioxidant enzyme activity, and elevated ROS levels in the leaves. The analysis of the gene expression profiles revealed that GA3 up-regulated the genes associated with disease resistance, such as WRKYs, NACs, MYBs, Cyt P450s, and ERFs, while it down-regulated the DELLA protein, a key agent in GA signaling. In contrast, PP333 induced gene expression changes that were the opposite of those caused by the GA3 treatment. These findings suggest that GA plays an essential role in the tomato's defense response against TYLCV and acts as a positive regulator of ROS scavenging and the expression of resistance-related genes.

Refining tomato disease recognition: hyperparameter tuning on ResNet-101 architecture for precise leaf-based classification

Tomatoes plants are widely recognized as versatile vegetables globally. This study aims to develop a high-precision web interface for classifying various leaf diseases in tomatoes. Utilizing a convolutional neural network (CNN) algorithm using the residual network-101 (ResNet-101) architecture, this tool aids farmers in accurately identifying leaf diseases in tomatoes, thereby reducing the risk of crop failure. The dataset comprises 6,800 images, categorized into four classes: early blight, spider mites two spotted, tomato yellow leaf curl virus, and healthy tomatoes, each containing 1,700 images. Hyperparameter tuning was conducted as part of an experiment aimed at enhancing the performance of the model. Experiments involved varying epoch values (10, 25, 30, 50, 60, 75, 100, and 110), a fixed batch size of 4, different learning rates (0.1, 0.01, 0.001, 0.0001), and num workers (4, 8, 16). The results demonstrated an accuracy of 99% with 100 epochs, a batch size of 4, a learning rate of 0.001, and 16 num workers. Consequently, this research contributes to a deeper understanding of disease management in tomato plants, ensuring optimal quality and quantity of the harvest.

Begomovirus Transmission to Tomato Plants Is Not Hampered by Plant Defenses Induced by Dicyphus hesperus Knight.

Plants can respond to insect infestation and virus infection by inducing plant defenses, generally mediated by phytohormones. Moreover, plant defenses alter host quality for insect vectors with consequences for the spread of viruses. In agricultural settings, other organisms commonly interact with plants, thereby inducing plant defenses that could affect plant-virus-vector interactions. For example, plant defenses induced by omnivorous insects can modulate insect behavior. This study focused on tomato yellow leaf curl virus (TYLCV), a plant virus of the family Geminiviridae and genus Begomovirus. It is transmitted in a persistent circulative manner by the whitefly Bemisia tabaci Gennadius (Hemiptera: Aleyrodidae), posing a global threat to tomato production. Mirids (Hemiptera: Miridae) are effective biological control agents of B. tabaci, but there is a possibility that their omnivorous nature could also interfere with the process of virus transmission. To test this hypothesis, this study first addressed to what extent the mirid bug Dicyphus hesperus Knight induces plant defenses in tomato. Subsequently, the impact of this plant-omnivore interaction on the transmission of TYLCV was evaluated. Controlled cage experiments were performed in a greenhouse setting to evaluate the impact of mirids on virus transmission and vector acquisition by B. tabaci. While we observed a reduced number of whiteflies settling on plants exposed to D. hesperus, the plant defenses induced by the mirid bug did not affect TYLCV transmission and accumulation. Additionally, whiteflies were able to acquire comparable amounts of TYLCV on mirid-exposed plants and control plants. Overall, the induction of plant defenses by D. hesperus did not influence TYLCV transmission by whiteflies on tomato.

Molecular validation of Tomato Yellow Leaf curl Virus (TYLCV) (Ty-1/3, Ty-2 and Ty-3), Fusarium wilt (I-2) and Root- Knot Nematode (Mi-1) resistance in the breeding lines of tomato (Solanum lycopersicum L.)

Molecular validation of Tomato Yellow Leaf curl Virus (TYLCV) (Ty-1/3, Ty-2 and Ty-3), Fusarium wilt (I-2) and Root- Knot Nematode (Mi-1) resistance in the breeding lines of tomato (Solanum lycopersicum L.)

Coat protein of a whitefly-vectored plant virus as a delivery system to target whitefly.

The sweet potato whitefly Bemisia tabaci (Hemiptera: Aleyrodidae) is responsible for significant crop losses and presents one of the greatest challenges for global agricultural pest management. Management of whitefly populations and associated plant viral diseases is hindered by widespread whitefly resistance to chemical insecticides. An alternative control approach involves the use of insect-specific neurotoxins, but these require delivery from the whitefly gut into the haemocoel. Here we demonstrate that the coat protein (CP) of a begomovirus, Tomato yellow leaf curl virus, is sufficient for delivery of fused proteins into the whitefly haemocoel without virion assembly. Following feeding on the recombinant CP-P-mCherry fusion (where -P- is a proline-rich linker), mCherry fluorescence was detected in the dorsal aorta and pericardial cells of the whitefly, but not in those of whitefly fed on negative control treatments, indicating effective CP-mediated delivery of mCherry into the whitefly haemocoel. Significant mortality was observed in whiteflies fed on a fusion of CP-P to the insect-specific neurotoxin Hv1a, but not in whiteflies fed on CP-P fused to a disarmed Hv1a mutant. Begomovirus coat protein - insect neurotoxin fusions hold considerable potential for transgenic resistance to whitefly providing valuable tools for whitefly management.

Commodity risk assessment of Petunia spp. and Calibrachoa spp. unrooted cuttings from Kenya.

The European Commission requested the EFSA Panel on Plant Health to evaluate the probability of entry of pests (likelihood of pest freedom at entry), including both regulated and non-regulated pests, associated with unrooted cuttings of the genera Petunia and Calibrachoa produced under physical isolation in Kenya. The relevance of any pest for this opinion was based on evidence following defined criteria, based on the methodology used for High-Risk Plants adapted for the specificity of this assessment. Fourteen EU-regulated pests (Bemisia tabaci, cowpea mild mottle virus, Liriomyza huidobrensis, Liriomyza sativae, Liriomyza trifolii, potato leafroll virus, potato spindle tuber viroid, Ralstonia pseudosolanacearum, R. solanacearum, Scirtothrips dorsalis, tomato mild mottle virus, tomato spotted wilt virus, tomato yellow leaf curl virus and Xanthomonas vesicatoria) and six EU non-regulated pests (Aleurodicus dispersus, pepper veinal mottle virus, Nipaecoccus viridis, Phenacoccus solenopsis, Tetranychus neocaledonicus and tomato yellow ring virus) fulfilled all relevant criteria and were selected for further evaluation. For these pests, the risk mitigation measures proposed in the technical dossier from Kenya were evaluated, taking into account the possible limiting factors. Additionally, an expert judgement is given on the likelihood of pest freedom, taking into consideration the risk mitigation measures acting on the pest, including uncertainties associated with the assessment. The estimated degree of pest freedom varies among the pests evaluated, with T. neocaledonicus being the pest most frequently expected on the imported cuttings. The Expert Knowledge Elicitation indicated, with 95% certainty, that between 9942 and 10,000 bags containing unrooted cuttings of Petunia spp. and Calibrachoa spp. per 10,000 would be free of T. neocaledonicus.

Natural occurrence of tomato chlorosis virus on tomatillo (Physalis philadelphica) in the United States.

Tomatillo (Physalis philadelphica L.) is an annual plant native to Mexico and Guatemala, and cultivated in other tropical and subtropical regions. In October 2023, tomatillo plants with interveinal yellowing of leaves, marginal chlorosis, leaf thickening, and leaf rolling symptoms (Figure 1) were observed at Colquitt and Tift County, Georgia, US. The disease incidence ranged from 80-100 % which reduced fruit quality and marketability. Twenty tomatillo leaves exhibiting severe symptoms were collected, and, sub-sampled of the leaves were pooled into microcentrifuge tubes. Further, MagMAX 96 viral RNA isolation kit (Thermo Fisher Scientific, US), was used for the extraction of (n=4) total nucleic acid (TNA) (Kavalappara et al. 2021). Symptomatic leaves were tested for the presence of insect-transmitted viruses such as begomovirus (tomato yellow leaf curl virus, TYLCV), potyvirus (turnip mosaic virus, TuMV), crinivirus (tomato infectious chlorosis virus, TICV; tomato chlorosis virus, ToCV), and tospovirus (orthotospovirus tomatomaculae, TSWV). Polymerase chain reaction (PCR) was performed for detecting TYLCV, using gene-specific primers (Kumar et al., 2023). However, for ToCV, TuMV and TICV detection, cDNA was prepared using 100 ng of TNA as a template, followed by the PCR ( Liu et al., 2012). Moreover, the detection of TSWV was conducted using immuno-strips (Adgia, US) following the manufacturer's instructions. ToCV was detected from all the tested samples, while TuMV, TICV, TYLCV and TSWV were not detected in any symptomatic tissues. In addition, RT-PCR was performed using gene-specific primers targeting the RNA-dependent RNA polymerase (RdRP) gene and the heat-shock protein 70 (Hsp70) gene of ToCV. The PCR amplicon of 439 bp encoding Hsp70 and 643 bp corresponding to RdRP was gel-purified and Sanger sequenced (Azenta Life Sciences, US). BLASTn analysis shows RdRP gene from ToCV-tomatillo (OR905600) has 100 % identity with ToCV of RNA1 segment (RdRP, GenBank accession no. AY903447, Florida, US), while Hsp70 gene (OR900219) has 100 % identity with ToCV of RNA2 segment (Hsp70, GenBank accession no. LC778246, Cairo, Egypt). In addition, the symptomatic tomatillo leaves were studied for transmission assay using tomato, employing non-viruliferous whiteflies (Bemisia tabaci) with 48 h of acquisition access period. Further, two weeks post-infection, the presence of ToCV was detected from the test plants while other whitefly-transmitted viruses remins undetected. In 2023, ToCV is widespread in tomato-growing counties, infecting commercially grown tomato cultivars with intermediate resistance against TYLCV-IL (Israel strain). However, tomatillo plants infected with TuMV in California (Liu et al., 2012), TSWV in Georgia, (Díaz-Pérez and Pappu 2000) and TYLCV in Mexico (Gámez-Jiménez et al. 2009) were reported. This study suggests that tomatillo could be a permissive host for ToCV while restrictive to other prevalent viruses in the region. A recent investigation speculates a potential synergistic interaction between ToCV and TYLCV-IL, exacerbating the breakdown of host resistance in tomato (Fiallo-Olivé et al. 2019, Kumar et al. 2023). To the best of our knowledge, this is the first report for the natural incidence of ToCV on tomatillo within the US. The findings will contribute to developing more effective management strategies against emerging viral threats.

Role of aminopeptidase N-like in the acquisition of begomoviruses by Bemisia tabaci, the whitefly vector.

Sri Lankan cassava mosaic virus (SLCMV) is a prominent causative agent of cassava mosaic disease in Asia and relies on the whitefly Bemisia tabaci cryptic complex for its transmission. However, the molecular mechanisms involved in SLCMV transmission by B.tabaci have yet to be understood. In this study, we identified an aminopeptidase N-like protein (BtAPN) in B.tabaci AsiaII1, an efficient vector of SLCMV, which is involved in the SLCMV transmission process. Through the use of glutathione S-transferase pull-down assay and LC-MS/MS analysis, we demonstrated the interaction between BtAPN and the coat protein (CP) of SLCMV. This interaction was further confirmed in vitro, and we observed an induction of BtAPN gene expression following SLCMV infection. By interfering with the function of BtAPN, the quantities of SLCMV were significantly reduced in various parts of B.tabaci AsiaII1, including the whole body, midgut, hemolymph, and primary salivary gland. Furthermore, we discovered that BtAPN is conserved in B.tabaci Middle East-Asia Minor 1 (MEAM1) and interacts with the CP of tomato yellow leaf curl virus (TYLCV), a begomovirus known to cause severe damage to tomato production. Blocking BtAPN with antibody led to a significant reduction in the quantities of TYLCV in whitefly whole body and organs/tissues. These results demonstrate that BtAPN plays a generic role in interacting with the CP of begomoviruses and positively regulates their acquisition by the whitefly.

D-Limonene Affects the Feeding Behavior and the Acquisition and Transmission of Tomato Yellow Leaf Curl Virus by Bemisia tabaci.

Bemisia tabaci (Gennadius) is an important invasive pest transmitting plant viruses that are maintained through a plant-insect-plant cycle. Tomato yellow leaf curl virus (TYLCV) can be transmitted in a persistent manner by B. tabaci, which causes great losses to global agricultural production. From an environmentally friendly, sustainable, and efficient point of view, in this study, we explored the function of d-limonene in reducing the acquisition and transmission of TYLCV by B. tabaci as a repellent volatile. D-limonene increased the duration of non-feeding waves and reduced the duration of phloem feeding in non-viruliferous and viruliferous whiteflies by the Electrical Penetration Graph technique (EPG). Additionally, after treatment with d-limonene, the acquisition and transmission rate of TYLCV was reduced. Furthermore, BtabOBP3 was determined as the molecular target for recognizing d-limonene by real-time quantitative PCR (RT-qPCR), fluorescence competitive binding assays, and molecular docking. These results confirmed that d-limonene is an important functional volatile which showed a potential contribution against viral infections with potential implications for developing effective TYLCV control strategies.

Transcriptome and small RNAome profiling uncovers how a recombinant begomovirus evades RDRγ-mediated silencing of viral genes and outcompetes its parental virus in mixed infection.

Tomato yellow leaf curl virus (TYLCV, genus Begomovirus, family Geminiviridae) causes severe disease of cultivated tomatoes. Geminiviruses replicate circular single-stranded genomic DNA via rolling-circle and recombination-dependent mechanisms, frequently generating recombinants in mixed infections. Circular double-stranded intermediates of replication also serve as templates for Pol II bidirectional transcription. IS76, a recombinant derivative of TYLCV with a short sequence in the bidirectional promoter/origin-of-replication region acquired from a related begomovirus, outcompetes TYLCV in mixed infection and breaks disease resistance in tomato Ty-1 cultivars. Ty-1 encodes a γ-clade RNA-dependent RNA polymerase (RDRγ) implicated in Dicer-like (DCL)-mediated biogenesis of small interfering (si)RNAs directing gene silencing. Here, we profiled transcriptome and small RNAome of Ty-1 resistant and control susceptible plants infected with TYLCV, IS76 or their combination at early and late infection stages. We found that RDRγ boosts production rates of 21, 22 and 24 nt siRNAs from entire genomes of both viruses and modulates DCL activities in favour of 22 and 24 nt siRNAs. Compared to parental TYLCV, IS76 undergoes faster transition to the infection stage favouring rightward transcription of silencing suppressor and coat protein genes, thereby evading RDRγ activity and facilitating its DNA accumulation in both single and mixed infections. In coinfected Ty-1 plants, IS76 efficiently competes for host replication and transcription machineries, thereby impairing TYLCV replication and transcription and forcing its elimination associated with further increased siRNA production. RDRγ is constitutively overexpressed in Ty-1 plants, which correlates with begomovirus resistance, while siRNA-generating DCLs (DCL2b/d, DCL3, DCL4) and genes implicated in siRNA amplification (α-clade RDR1) and function (Argonaute2) are upregulated to similar levels in TYLCV- and IS76-infected susceptible plants. Collectively, IS76 recombination facilitates replication and promotes expression of silencing suppressor and coat proteins, which allows the recombinant virus to evade the negative impact of RDRγ-boosted production of viral siRNAs directing transcriptional and posttranscriptional silencing.

Systemic resistance induced in tomato plants by Beauveria bassiana-derived proteins against tomato yellow leaf curl virus and aphid Myzus persicae.

Tomato (Solanum lycopersicum L.) is an economically important vegetable crop around the globe. Tomato yellow leaf curling (TYLC) is the most devastating viral disease posing a serious threat to tomato production throughout the tropical and subtropical world. Induction of microbe-mediated systemic resistance in plants has been of great interest in recent years as a novel microbiological tool in disease and insect pest management. This in-vitro study aimed to determine the effectiveness of different strains (BB252, BB72 and ARSEF-2860) of a hypocreal fungus Beauveria bassiana against TYLCV disease and aphid Myzus persicae. Potted tomato plants exogenously treated with conidial and filtrate suspensions of B. bassiana strains and of their partially purified or purified proteins were exposed to TYLCV inoculum and aphid M. persicae. Results showed a significant suppression of TYLCV disease severity index by the exogenous application of conidial, filtrate and protein treatments of all B. bassiana strains and this response was directly proportional to the treatment concentration. Similarly, mean fecundity rate of M. persicae was also significantly reduced by the highest concentration of ARSEF-2860-derived elicitor protein PeBb1, followed by the highest concentrations of BB252- and BB72-derived partially purified proteins. Moreover, these B. bassiana-derived proteins also caused a significant upregulation of most of the plant immune marker genes associated with plant defense. Overall, the study findings suggest that these B. bassiana strains and their partially purified or purified elicitor proteins could be effective biological tools for the management of TYLCV and aphid infestation on tomato plants. © 2023 Society of Chemical Industry.

Variability in indigenous Pakistani tomato lines and worldwide reference collection for Tomato Mosaic Virus (ToMV) and Tomato Yellow Leaf Curl Virus (TYLCV) infection.

Local and exotic germplasm of tomato remains a major source for genetic improvement. Assessment of such lines for biotic stresses particularly viral diseases are the most important criteria for selection in Pakistan, where Tomato Yellow Leaf Curl Virus (TYLCV) and Tomato Mosaic Virus (ToMV) are the major diseases/viruses. A set of 40 accessions (including indigenous Pakistani lines and exotic germplasm from Europe, the United States, and Asia) were evaluated for their resistance/infection response to ToMV with artificial inoculation under greenhouse conditions. Infection response was quantified through disease scoring and DAS-ELISA test (for ToMV). A subset of 24 lines, was further screened for TYLCV using disease scoring and TAS-ELISA. The tested lines showed significant variability for resistance to ToMV. Only one accession (Acc-17878) was resistant to the ToMV whereas seven accessions i.e. Acc-17890, AVR-261, CLN-312, AVR-321, EUR-333, CLN-352, and CLN-362 expressed resistance to TYLCV. Correlation between phenotypic evaluation was confirmed by the ELISA results in both diseases, although both tools complemented to assess the viral infection status. In future, tomato breeding programs must consider breeding for ToMV and TYLCV resistance (using identified germplasm in our study) so as to deliver virus resistant tomato varieties.

Breakdown of Ty-1-Based Resistance to Tomato Yellow Leaf Curl Virus in Tomato Plants at High Temperatures.

The global dissemination of the Israel (IL) and mild (Mld) strains of tomato yellow leaf curl virus (TYLCV) (family Geminiviridae, genus Begomovirus) is a major threat to tomato production in many regions worldwide. The use of resistant hybrid cultivars bearing the dominant resistance genes Ty-1, Ty-3, and Ty-3a has become a common practice for controlling tomato yellow leaf curl disease (TYLCD) caused by TYLCV. However, TYLCD symptoms have been sporadically observed in resistant cultivars grown in seasons when temperatures are high. In this study, we used TYLCV-resistant cultivars with confirmed presence of Ty-1, which were determined using newly developed allele-specific markers based on polymorphisms within the locus. These Ty-1-bearing resistant tomato plants and susceptible plants were infected with TYLCV and grown at moderate or high temperatures. Under high-temperature conditions, the Ty-1-bearing tomato cultivar Momotaro Hope (MH) infected with TYLCV-IL had severe TYLCD symptoms, which were almost equivalent to those of the susceptible cultivar. However, MH plants infected with TYLCV-Mld were symptomless or had slight symptoms under the same temperature condition. The quantitative analysis of the TYLCV-IL viral DNA content revealed a correlation between symptom development and viral DNA accumulation. Furthermore, under high-temperature conditions, TYLCV-IL caused severe symptoms in multiple commercial tomato cultivars with different genetic backgrounds. Our study provided the scientific evidence for the experientially known phenomenon by tomato growers, and it is anticipated that global warming, associated with climate change, could potentially disrupt the management of TYLCV in tomato plants mediated by the Ty-1 gene.

Commodity risk assessment of Petunia spp. and Calibrachoa spp. unrooted cuttings from Guatemala.

The European Commission requested the EFSA Panel on Plant Health to evaluate the probability of entry of pests (likelihood of pest freedom at entry), including both, regulated and non-regulated pests, associated with unrooted cuttings of the genera Petunia and Calibrachoa produced under physical isolation in Guatemala. The relevance of any pest for this opinion was based on evidence following defined criteria, based on the methodology used for high-risk plants adapted for the specificity of this assessment. Nineteen EU regulated pests (Bemisia tabaci, pepper golden mosaic virus, pepper huasteco yellow vein virus, tomato severe leaf curl virus, tomato yellow leaf curl virus, tomato spotted wilt virus, Liriomyza huidobrensis, Liriomyza sativae, Liriomyza trifolii, Bactericera cockerelli, Eotetranichus lewisi, Epitrix subcrinita, Epitrix cucumeris, Helicoverpa zea, Chloridea virescens, Spodoptera ornithogalli, Ralstonia solanacearum, Ralstonia pseudosolanacearum, Xanthomonas vesicatoria) and one EU non-regulated (Phenacoccus solenopsis) pest fulfilled all relevant criteria and were selected for further evaluation. For these pests, the risk mitigation measures proposed in the technical dossier from Guatemala were evaluated taking into account the possible limiting factors, and an expert judgement is given on the likelihood of pest freedom taking into consideration the risk mitigation measures acting on the pest, including uncertainties associated with the assessment. The limited and partially conflicting information provided in the dossier contributes to the wide estimates of pest freedom. The estimated degree of pest freedom varies among the pests evaluated, with Ralstonia spp. (R. solanacearum and R. pseudosolanacearum) being the pest most frequently expected on the imported cuttings. The expert knowledge elicitation indicated, with 95% certainty, that between 9916 and 10,000 bags containing unrooted cuttings per 10,000 would be free of Ralstonia spp.

Study of some physiological changes during the combined effects of TYLCV and drought stresses in a local tomato (Solanum lycopersicum L.) genotype

Global warming and drought, which are considered the main problems of our time, seriously damage the rural economy. Long-term drought manifests itself prominently in plant-pathogen relationships. The Vatan genotype infected with the tomato yellow leaf curl virus (TYLCV) showed tolerance to drought. Complex host-pathogen interactions are the basis of tomato plant response to combined biotic and abiotic stresses. According to the experiments, the tomato plant infected with TYLCV saves the plant from being destroyed by the pathogen during extreme drought, which helps to prevent crop loss. The amount of dry biomass in the leaves of the tomato plant increased during virus infection. The rate of RWC decreased in TYLCV-infected leaves. The content of photosynthetic pigments leads to a decrease in stressed tomatoes. Considering these, crop loss can be prevented by using plants resistant to TYLCV in seasons of water deficit, which is very important from an economic point of view. Keywords: Solanum lycopersicum L., drought, tomato yellow leaf curl virus, Bemisia tabaci, host plant-virus interaction, combined stress

Characterization of tomato lines for yield components, processing traits and disease resistance based on phenotype and molecular markers

Development of improved line is one of the major activities in hybrid tomato breeding. The aim of this study was to identify improved lines based on horticultural traits, total soluble solid (TSS) and diseases resistance particularly tomato yellow leaf curl virus (ToLCV). The genotypes consisted of improved lines, jointless tomato, cherry tomato and beta carotene rich lines. These lines were evaluated in field experiment and screened against ToLCV resistance under natural field condition for two years. Molecular markers based assays were performed for resistance genes to ToLCV (Ty-2 and Ty-3), late blight (Ph-3) and root knot nematode (Mi1-2). Based on fruit yield, promising genotypes were improved lines (VRT-06, VRT-19, VRT-34 and VRT-51), jointless (EC-605037 and EC-695037), cherry tomato (VRCRT-5), and beta-carotene line (KB-20). High TSS was noticed in improved line VRT-67 (5.36 °Brix), jointless EC-695037 (5.37 °Brix) and cherry tomato VRCYT-3 (8.22 °Brix). High ToLCV resistance was recorded in improved lines (VRT-06, VRT-19, VRT-30, VRT-34, VRT-50, VRT-51, and VRT-67), jointless (EC-695037, EC-605037, EC-605094, KB-Jointless and New-Jointless), cherry tomato (VRCYT-3, VRCYT-5, VRCYT-9 and VRCYT-15), and beta carotene (KB-2, KB-3-1, KB-3-2, KB-5, KB-10, KB-14, KB-17 and KB-20). Tomato line VRT-02 having dwarf plant type was found suitable for pot culture, whereas line H-88-78-2 had very delayed and partial fruit ripening. Thus, our study identified promising lines for breeding application for economically important horticultural traits.

Viruses and viroids in tomato (Solanum lycopersicum) and plant growth promoting rhizobacteria as a management alternative

Viruses and viroids cause several diseases in tomato (Solanum lycopersicum) worldwide, generating important economic losses. About 312 viruses and seven viroids have been associated, of which more than 28 are present in Mexico. Therefore, the use of Plant Growth-Promoting Rhizobacteria (PGPR) can be an effective alternative for the management of viruses and viroids. The genera Pseudomonas, Bacillus, Azospirillum, Anabena and Stenotrophomonas have been implemented against main viruses reported in tomato: Cucumber mosaic virus (CMV), Tobacco mosaic virus (TMV), Tomato chlorotic spot virus (TCSV), Tomato mottle virus (ToMoV), Tomato spotted wilt virus (TSWV), Tomato yellow leaf curl virus (TYLCV), Potato virus Y (PVY), Groundnut bud necrosis virus (GBNV), with benefits in decreased incidence and severity up to 80 % and yield increase over 40 %. In Mexico, only Bacillus has been used. The use of PGPR is a strategy that could mitigate the impact of viral and viroid diseases and can be integrated into integrated management.

Exploration of DNA Binding Proteins As a Versatile Tool for Fabrication of DNA-Protein Complexes and Its Application to Biosensing System

Introduction DNA-protein complexes composed of a DNA aptamer and an enzyme are useful to realize innovative biosensing systems; however, the stoichiometric and efficient fabrication method for DNA-protein complexes is yet to be developed. Chemical modification using amino acids on the surface of the protein is known as one irreversible conjugation way to fabricate DNA-protein complexes, but stoichiometric conjugation and retention of protein function to ensure high detection sensitivity are still a challenge. Therefore, in recent years, DNA binding proteins that covalently bind to specific DNA sequences have been utilized to fabricate DNA-protein complexes and applied as sensing elements. However, the binding efficiency to DNA of the DNA binding protein was low, which caused a decrease in detection sensitivity.Here, we introduce our challenge to utilize DNA binding proteins with high DNA binding ability as a versatile tool suitable for the fabrication of DNA-protein complexes. By fusing such DNA binding proteins to protein of interest, can be covalently linked to the fusion protein, leading to the preparation of stoichiometric DNA-protein complexes universally and conveniently (Fig. 1). We demonstrated the feasibility to construct a biosensing system of hemoglobin (Hb), composed of glucose oxidase (GOx) and anti-Hb aptamer as a representative example. Results & Discussion We explored the DNA binding proteins that can covalently bind to specific DNA sequences using the database to fabricate the stoichiometric and stable DNA-protein complex. Seventeen candidates were selected and classified into two families: HUH-endonuclease and uracil DNA glycosylase. We then selected three kinds of HUH-endonucleases: Rep domain of tomato yellow leaf curl virus (TYLCV), replication protein B (RepB), and conjugation transfer relaxase/helicase TraI (TraI). We expected them to have high recombinant production in the E. coli expression system because TYLCV and RepB have a relatively low molecular weight and TraI is derived from E. coli. We also selected UdgX because it is the only uracil DNA glycosylase known to form covalent bonds with DNA after removing uracil that aberrantly exists on DNA1). Thus, four DNA binding proteins with the ability to covalently bind to DNA were selected for further characterization.The binding ability of the four DNA binding proteins was compared. Each DNA binding protein was reacted with single stranded DNA (ssDNA) containing the corresponding recognition sequence and subjected to SDS-PAGE analysis. An ssDNA-dependent concentration band shift to higher molecular weight was observed for all DNA binding proteins upon covalent binding to each ssDNA. Based on the band quantification, we confirmed that UdgX had the highest binding efficiency and thus we used UdgX in following experiments.We attempted to detect Hb utilizing a biosensing element composed of GOx and anti-Hb aptamer fabricated using UdgX. We recently successfully integrated a protein coupling module, SpyCatcher/Tag system, into the universal fabrication method of sensing elements2-4). GOx and UdgX were also conjugated using SpyCatcher/SpyTag system, and then reacted with anti-Hb aptamer to fabricate aptamer-GOx complex. Hb has peroxidase activity and catalyzes chemiluminescence of luminol. Therefore, by using the aptamer-GOx complex, hydrogen peroxide produced by GOx is supplied to the Hb efficiently, and chemiluminescence of luminol is observed (Fig. 2).We confirmed homogeneous preparation of the aptamer-GOx complex by SDS-PAGE, and also the binding ability of aptamer and enzymatic activity of GOx. Using thus prepared DNA-protein complex, Hb was successfully detected with a high linearity in buffer and also in human serum without any washing procedures. The detection range was 4.0×10-3 to 32×10-3 g/L, this can meet the clinically required one (70 to 200 g/L) by diluting an actual sample. Because this system uses redox enzyme, it can be applied into the electrochemical measurement to achieve more rapid Hb detection. In this system, owing to integration of DNA binding protein and SpyCatcher/SpyTag system into sensing elements, a set of DNA aptamer and enzyme can be also easily exchanged, thus which has potential of various applications. References 1) P. B. Sang et al., Nucleic Acids Res., 43, 8452–8463 (2015).2) H. Kimura et al., Anal. Chem., 90, 14500-14506 (2018).3) H. Kimura et al., Biosens. Bioelectron., 175, 112885 (2021).4) D. Miura et al., Talanta, 234, 122638 (2021). Figure 1

Transcriptional and epigenetic changes during tomato yellow leaf curl virus infection in tomato

BackgroundGeminiviruses are DNA plant viruses that cause highly damaging diseases affecting crops worldwide. During the infection, geminiviruses hijack cellular processes, suppress plant defenses, and cause a massive reprogramming of the infected cells leading to major changes in the whole plant homeostasis. The advances in sequencing technologies allow the simultaneous analysis of multiple aspects of viral infection at a large scale, generating new insights into the molecular mechanisms underlying plant-virus interactions. However, an integrative study of the changes in the host transcriptome, small RNA profile and methylome during a geminivirus infection has not been performed yet. Using a time-scale approach, we aim to decipher the gene regulation in tomato in response to the infection with the geminivirus, tomato yellow leaf curl virus (TYLCV).ResultsWe showed that tomato undergoes substantial transcriptional and post-transcriptional changes upon TYLCV infection and identified the main altered regulatory pathways. Interestingly, although the principal plant defense-related processes, gene silencing and the immune response were induced, this cannot prevent the establishment of the infection. Moreover, we identified extra- and intracellular immune receptors as targets for the deregulated microRNAs (miRNAs) and established a network for those that also produced phased secondary small interfering RNAs (phasiRNAs). On the other hand, there were no significant genome-wide changes in tomato methylome at 14 days post infection, the time point at which the symptoms were general, and the amount of viral DNA had reached its maximum level, but we were able to identify differentially methylated regions that could be involved in the transcriptional regulation of some of the differentially expressed genes.ConclusionWe have conducted a comprehensive and reliable study on the changes at transcriptional, post-transcriptional and epigenetic levels in tomato throughout TYLCV infection. The generated genomic information is substantial for understanding the genetic, molecular and physiological changes caused by TYLCV infection in tomato.