Tomato yellow leaf curl virus: Characteristics, influence, and regulation mechanism.

Tomato yellow leaf curl virus (TYLCV), a DNA virus belonging to the genus Begomovirus, significantly impedes the growth and development of numerous host plants, including tomatoes and peppers. Due to its rapid mutation rate and frequent recombination events, achieving complete control of TYLCV proves exceptionally challenging. Consequently, identifying resistance mechanisms become crucial for safeguarding host plants from TYLCV-induced damage. This review article delves into the global distribution, dispersal patterns, and defining characteristics of TYLCV. Moreover, the intricate interplay between TYLCV and various influencing factors, such as insect vectors, susceptible host plants, and abiotic stresses, plays a pivotal role in plant-TYLCV interactions. The review offers an updated perspective on recent investigations focused on plant response mechanisms to TYLCV infection, including the intricate relationship between TYLCV, whiteflies, and regulatory factors. This comprehensive analysis aims to establish a foundation for future research endeavors exploring the molecular mechanisms underlying TYLCV infection and the development of plant resistance through breeding programs.

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.

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.

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 Asia II 1, 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 Asia II 1, 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.

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.

Phenotype and signaling pathway analysis to explore the interaction between tomato plants and TYLCV in different organs.

Tomato yellow leaf curl disease (TYLCD), caused by Tomato yellow leaf curl virus (TYLCV), is one of the most destructive diseases in tomato cultivation. By comparing the phenotypic characteristics and virus quantities in the susceptible variety 'Cooperation 909 Red Tomatoes' and the resistant variety 'Huamei 204' after inoculation with TYLCV infectious clones, our study discovered that the root, stem and leaf growth of the susceptible variety 'Cooperation 909 Red Tomatoes' were severely hindered and the resistant variety 'Huamei 204' showed growth inhibition only in roots. TYLCV accumulation in roots were significantly higher than in leaves. Further, we examined the expression of key genes in the SA and JA signalling pathways in leaves, stems and roots and found the up-regulation of SA-signalling genes in all organs of the susceptible variety after inoculation with TYLCV clones. Interestingly, SlJAZ2 in roots of the resistant variety was significantly down-regulated upon TYLCV infection. Further, we silenced the SlNPR1 and SlCOI1 genes individually using virus induced gene silencing system in tomato plants. We found that viruses accumulated to a higher level in SlNPR1 silenced plants than wild type plants, and the virus quantity in roots was significantly increased in SlCOI1 silenced plants. These results provide new insights for advancing research in understanding tomato-TYLCV interaction.

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

BACKGROUND: Geminiviruses 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). RESULTS: We 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. CONCLUSION: We 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.

Genome-wide identification and expression analysis of wall-associated kinase (WAK) and WAK-like kinase gene family in response to tomato yellow leaf curl virus infection in Nicotiana benthamiana.

BACKGROUND: Tomato yellow leaf curl virus (TYLCV) is a major monopartite virus in the family Geminiviridae and has caused severe yield losses in tomato and tobacco planting areas worldwide. Wall-associated kinases (WAKs) and WAK-like kinases (WAKLs) are a subfamily of the receptor-like kinase family implicated in cell wall signaling and transmitting extracellular signals to the cytoplasm, thereby regulating plant growth and development and resistance to abiotic and biotic stresses. Recently, many studies on WAK/WAKL family genes have been performed in various plants under different stresses; however, identification and functional survey of the WAK/WAKL gene family of Nicotiana benthamiana have not yet been performed, even though its genome has been sequenced for several years. Therefore, in this study, we aimed to identify the WAK/WAKL gene family in N. benthamiana and explore their possible functions in response to TYLCV infection. RESULTS: Thirty-eight putative WAK/WAKL genes were identified and named according to their locations in N. benthamiana. Phylogenetic analysis showed that NbWAK/WAKLs are clustered into five groups. The protein motifs and gene structure compositions of NbWAK/WAKLs appear to be highly conserved among the phylogenetic groups. Numerous cis-acting elements involved in phytohormone and/or stress responses were detected in the promoter regions of NbWAK/WAKLs. Moreover, gene expression analysis revealed that most of the NbWAK/WAKLs are expressed in at least one of the examined tissues, suggesting their possible roles in regulating the growth and development of plants. Virus-induced gene silencing and quantitative PCR analyses demonstrated that NbWAK/WAKLs are implicated in regulating the response of N. benthamiana to TYLCV, ten of which were dramatically upregulated in locally or systemically infected leaves of N. benthamiana following TYLCV infection. CONCLUSIONS: Our study lays an essential base for the further exploration of the potential functions of NbWAK/WAKLs in plant growth and development and response to viral infections in N. benthamiana.

The transcriptional regulator JAZ8 interacts with the C2 protein from geminiviruses and limits the geminiviral infection in Arabidopsis.

Jasmonates (JAs) are phytohormones that finely regulate critical biological processes, including plant development and defense. JASMONATE ZIM-DOMAIN (JAZ) proteins are crucial transcriptional regulators that keep JA-responsive genes in a repressed state. In the presence of JA-Ile, JAZ repressors are ubiquitinated and targeted for degradation by the ubiquitin/proteasome system, allowing the activation of downstream transcription factors and, consequently, the induction of JA-responsive genes. A growing body of evidence has shown that JA signaling is crucial in defending against plant viruses and their insect vectors. Here, we describe the interaction of C2 proteins from two tomato-infecting geminiviruses from the genus Begomovirus, tomato yellow leaf curl virus (TYLCV) and tomato yellow curl Sardinia virus (TYLCSaV), with the transcriptional repressor JAZ8 from Arabidopsis thaliana and its closest orthologue in tomato, SlJAZ9. Both JAZ and C2 proteins colocalize in the nucleus, forming discrete nuclear speckles. Overexpression of JAZ8 did not lead to altered responses to TYLCV infection in Arabidopsis; however, knock-down of JAZ8 favors geminiviral infection. Low levels of JAZ8 likely affect the viral infection specifically, since JAZ8-silenced plants neither display obvious developmental phenotypes nor present differences in their interaction with the viral insect vector. In summary, our results show that the geminivirus-encoded C2 interacts with JAZ8 in the nucleus, and suggest that this plant protein exerts an anti-geminiviral effect.

Hrip1 enhances tomato resistance to yellow leaf curl virus by manipulating the phenylpropanoid biosynthesis and plant hormone pathway.

Tomato yellow leaf curl virus (TYLCV) causes tremendous losses of tomato worldwide. An elicitor Hrip1, which produced by Alternaria tenuissima, can serve as a pathogen-associated molecular patterns (PAMPs) to trigger the immune defense response in Nicotiana benthamiana. Here, we show that Hrip1 can be targeted to the extracellular space and significantly delayed the development of symptoms caused by TYLCV in tomato. In basis of RNA-seq profiling, we find that 1621 differential expression genes (DEGs) with the opposite expression patterns are enriched in plant response to biotic stress between Hrip1 treatment and TYLCV infection of tomato. Thirty-two known differential expression miRNAs with the opposite expression patterns are identified by small RNA sequencing and the target genes of these miRNAs are significantly enriched in phenylpropanoid biosynthesis, plant hormone signal transduction and peroxisome. Based on the Pearson correlation analysis, 13 negative and 21 positive correlations are observed between differential expression miRNAs and DEGs. These miRNAs, which act as a key mediator of tomato resistance to TYLCV induced by Hrip1, regulate the expression of phenylpropanoid biosynthesis and plant hormone signal transduction-related genes. Taken together, our results provide an insight into tomato resistance to TYLCV induced by PAMP at transcriptional and posttranscriptional levels. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03426-6.

Expanding growers' choice of plant disease management options can promote suboptimal social outcomes.

Previous models of growers' decision-making during epidemics have unrealistically limited disease management choices to just two options. Here, we expand previous game-theoretic models of grower decision-making to include three control options: a crop that is tolerant, resistant or susceptible to disease. Using tomato yellow leaf curl virus (TYLCV) as a case study, we investigate how growers can be incentivized to use different control options to achieve socially optimal outcomes. To do this, we consider the efforts of a 'social planner' who moderates the price of crops. We find that subsidizing a tolerant crop costs the social planner more in subsidies, as its use encourages selfishness and widespread adoption. Subsidizing a resistant crop, however, provides widespread benefits by reducing the prevalence of disease across the community of growers, including those that do not control, reducing the number of subsidies required from the social planner. We then use Gini coefficients to measure equitability of each subsidization scheme. This study highlights how grower behaviour can be altered using crop subsidies to promote socially optimal outcomes during epidemics.

Silencing of SlDRB1 gene reduces resistance to tomato yellow leaf curl virus (TYLCV) in tomato (Solanum lycopersicum).

Double-stranded RNA-binding proteins are small molecules in the RNA interference (RNAi) pathway that form the RNAi machinery together with the Dicer-like protein (DCL) as a cofactor. This machinery cuts double-stranded RNA (dsRNA) to form multiple small interfering RNAs (siRNAs). Our goal was to clarify the function of DRB in tomato resistant to TYLCV. In this experiment, the expression of the SlDRB1 and SlDRB4 genes was analyzed in tomato leaves by qPCR, and the function of SlDRB1 and SlDRB4 in resistance to TYLCV was investigated by virus-induced gene silencing (VIGS). Then, peroxidase activity was determined. The results showed that the expression of SlDRB1 gradually increased after inoculation of 'dwarf tomato' plants with tomato yellow leaf curl virus (TYLCV), but this gene was suppressed after 28 days. Resistance to TYLCV was significantly weakened after silencing of the SlDRB1 gene. However, there were no significant expression differences in SlDRB4 after TYLCV inoculation. Our study showed that silencing SlDRB1 attenuated the ability of tomato plants to resist virus infection; therefore, SlDRB1 may play a key role in the defense against TYLCV in tomato plants, whereas SlDRB4 is likely not involved in this defense response. Taken together, These results suggest that the DRB gene is involved in the mechanism of antiviral activity.

The V2 Protein from the Geminivirus Tomato Yellow Leaf Curl Virus Largely Associates to the Endoplasmic Reticulum and Promotes the Accumulation of the Viral C4 Protein in a Silencing Suppression-Independent Manner.

Viruses are strict intracellular parasites that rely on the proteins encoded in their genomes for the effective manipulation of the infected cell that ultimately enables a successful infection. Viral proteins have to be produced during the cell invasion and takeover in sufficient amounts and in a timely manner. Silencing suppressor proteins evolved by plant viruses can boost the production of viral proteins; although, additional mechanisms for the regulation of viral protein production likely exist. The strongest silencing suppressor encoded by the geminivirus tomato yellow leaf curl virus (TYLCV) is V2: V2 suppresses both post-transcriptional and transcriptional gene silencing (PTGS and TGS), activities that are associated with its localization in punctate cytoplasmic structures and in the nucleus, respectively. However, V2 has been previously described to largely localize in the endoplasmic reticulum (ER), although the biological relevance of this distribution remains mysterious. Here, we confirm the association of V2 to the ER in Nicotiana benthamiana and assess the silencing suppression activity-independent impact of V2 on protein accumulation. Our results indicate that V2 has no obvious influence on the localization of ER-synthesized receptor-like kinases (RLKs) or ER quality control (ERQC)/ER-associated degradation (ERAD), but dramatically enhances the accumulation of the viral C4 protein, which is co-translationally myristoylated, possibly in proximity to the ER. By using the previously described V2C84S/86S mutant, in which the silencing suppression activity is abolished, we uncouple RNA silencing from the observed effect. Therefore, this work uncovers a novel function of V2, independent of its capacity to suppress silencing, in the promotion of the accumulation of another crucial viral protein.

Physiological and transcriptomic analyses revealed gene networks involved in heightened resistance against tomato yellow leaf curl virus infection in salicylic acid and jasmonic acid treated tomato plants.

Tomato yellow leaf curl virus (TYLCV), a member of the genus Begomovirus of the Geminiviridae family, causes leaf curl disease of tomato that significantly affects tomato production worldwide. SA (salicylic acid), JA (jasmonic acid) or the JA mimetic, COR (coronatine) applied exogenously resulted in improved tomato resistance against TYLCV infection. When compared to mock treated tomato leaves, pretreatment with the three compounds followed by TYCLV stem infiltration also caused a greater accumulation of H2O2. We employed RNA-Seq (RNA sequencing) to identify DEGs (differentially expressed genes) induced by SA, JA, COR pre-treatments after Agro-inoculation of TYLCV in tomato. To obtain functional information on these DEGs, we annotated genes using gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) databases. Based on our comparative analysis, differentially expressed genes related to cell wall metabolism, hormone signaling and secondary metabolism pathways were analyzed in compound treated samples. We also found that TYLCV levels were affected in SlNPR1 and SlCOI1 silenced plants. Interestingly, compared to the mock treated samples, SA signaling was hyper-activated in SlCOI1 silenced plants which resulted in a significant reduction in viral titer, whereas in SINPR1 silencing tomato plants, there was a 19-fold increase in viral load. Our results indicated that SA, JA, and COR had multiple impacts on defense modulation at the early stage of TYLCV infection. These results will help us better understand SA and JA induced defenses against viral invasion and provide a theoretical basis for breeding viral resistance into commercial tomato accessions.

Exogenous Nitric Oxide Alleviates the Damage Caused by Tomato Yellow Leaf Curl Virus in Tomato through Regulation of Peptidase Inhibitor Genes.

The tomato yellow leaf curl virus (TYLCV) is the causal agent of one of the most severe diseases affecting tomato growth; however, nitric oxide (NO) can mediate plant resistance. This study investigated the molecular mechanism of exogenous NO donor-mediated disease resistance in tomato seedlings. Tomato seedlings were treated with sodium nitroprusside and TYLCV and subjected to phenotypic, transcriptomic, and physiological analyses. The results show that exogenous NO significantly reduced disease index, MDA content, and virus content (71.4%), significantly increased stem length and fresh weight of diseased plants (p < 0.05), and improved photosynthesis with an induction effect of up to 44.0%. In this study, it was found that the reduction in virus content caused by the increased expression of peptidase inhibitor genes was the main reason for the increased resistance in tomatoes. The peptidase inhibitor inhibited protease activity and restrained virus synthesis, while the significant reduction in virus content inevitably caused a partial weakening or shutdown of the disease response process in the diseased plant. In addition, exogenous NO also induces superoxide dismutase, peroxidase activity, fatty acid elongation, resistance protein, lignin, and monoterpene synthesis to improve resistance. In summary, exogenous NO enhances resistance in tomatoes mainly by regulating peptidase inhibitor genes.

Development of a Novel Tissue Blot Hybridization Chain Reaction for the Identification of Plant Viruses.

Assays for the high throughput screening of crops for virus monitoring need to be quick, easy, and low cost. One method involves using tissue blot immunoassays (TBIA), where plant stems are blotted onto nitrocellulose membrane and screened with available antibodies against a range of viruses. TBIAs are inexpensive but limited by antibody availability and specificity. To circumvent the antibody limitations, we developed the tissue blot hybridization chain reaction (TB-HCR). As with TBIA, plant stems are blotted onto a nitrocellulose membrane, however, TB-HCR involves using nucleic acid probes instead of antibodies. We demonstrated for the first time that TB-HCR can be used for plant viruses by designing and testing probes against species from several virus genera including begomovirus, polerovirus, luteovirus, cucumovirus, and alfamovirus. We also explored different hairpin reporter methods such as biotin/streptavidin-AP and the Alexa Fluor-488 Fluorophore. TB-HCR has applications for low-cost diagnostics for large sample numbers, rapid diagnostic deployment for new viruses, and can be performed as a preliminary triage assay prior to downstream applications.

The Establishment and Spread of a Newly Introduced Begomovirus in a Dry Tropical Environment Using Tomato Yellow Leaf Curl Virus as a Case Study.

Early detection of tomato yellow leaf curl virus (TYLCV) in a previously unaffected tomato production district in Australia allowed its spread to be evaluated spatially and temporally. The population dynamics of the TYLCV vector, Bemisia argentifolii (silverleaf whitefly, SLW), were also evaluated. The district is a dry tropical environment with a clear break to commercial production during the summer wet season. The incidence of TYLCV within crops and its prevalence through the district was influenced by weather, location, vector movements, and the use of Ty-1 virus-resistant hybrids. Rainfall had an important influence, with late summer and early autumn rain suppressing the levels of SLW and, by contrast, a dry summer supporting faster population growth. The use of Ty-1 hybrids appears to have reduced the incidence of TYLCV in this district. There was limited use of Ty-1 hybrids during 2013, and by season end, crops had moderate levels of SLW and high virus incidence. The 2015 and early 2016 season had high SLW populations, but TYLCV incidence was lower than in 2013, possibly due to the widespread adoption of the Ty-1 hybrids reducing virus spread. This study provides valuable epidemiology data for future incursions of begomoviruses, and other viruses spread by SLW.

Structural basis of DNA recognition of tomato yellow leaf curl virus replication-associated protein.

Conserved and multifunctional Geminivirus Replication-associated Protein (Rep) specifically recognizes the replication origin and initiates viral DNA replication. We report the X-ray crystallography-based structures of two complexes containing the N-terminal domain (5-117aa) of Tomato yellow leaf curl virus (TYLCV) Rep: the catalytically-dead Rep in complex with nonanucleotide ssDNA (Rep5-117 Y101F-ssDNA) as well as the catalytically-active phosphotyrosine covalent adduct (Rep5-117-ssDNA). These structures provide functional insight into the role of Rep in viral replication. Metal ions stabilize the DNA conformation by interacting with the phosphate group of adenine and thus promote formation of the catalytic center. Furthermore, we identified a compound that inhibits the binding of Rep to ssDNA and dsDNA and found that the addition of metal ions compromises the inhibitory effectiveness of this compound. This study demonstrates the mechanism of DNA recognition and cleavage process of viral Rep, emphasizing the role of metal ions.

A multiplex RT-PCR assay combined with co-extraction of DNA and RNA for simultaneous detection of TYLCV and ToCV in whitefly.

Tomato yellow leaf curl virus (TYLCV) and tomato chlorosis virus (ToCV) were transmitted by the sweet potato whitefly Bemisia tabaci (Gennadius) and cause serious yield losses on tomato around the world. To understand the actual situation of co-infection of TYLCV and ToCV of individual whiteflies, we developed multiplex RT-PCR combined with co-extraction of DNA and RNA from a single whitefly. First, a nucleic acid extraction method previously reported was modified and adopted to obtain the RNA-DNA mixture including TYLCV and ToCV in a simple form without manual homogenization. Second, primers were newly designed in actin gene of B. tabaci for the confirmation of extraction and PCR success, and multiplex RT-PCR method was developed using specific primer sets for TYLCV, ToCV and B. tabaci. This method enables the detection of TYLCV and ToCV from a single insect and efficient use of field samples obtained using sticky traps. The method will be useful to monitor infection status of TYLCV and ToCV in the field while reducing labor and cost.